RIOLOfiY  LIRRAUL 


THE  EXPERIMENTAL  PRODUCTION  OF .:  MACROPHAGES  IN 
THE  CIRCULATING  BtOOD  * 

MIRIAM  E.  SIMPSON 
(From  the  Anatomical  Laboratory  of  the  University  of  California 

The  great  significance  of  certain  large  phagocytic  cells  in 
the  metabolic  and  protective  reactions  of  the  body  is  just 
beginning  to  be  known.  These  cells  have  been  variously  called 
pyrrhol  cells  (Goldman),  adventitia  cells  (Marchand),  rhagio- 
crine  cells  (Renaut),  resting  wandering  cells  (Maximo w), 
endothelial  leucocytes  (Mallory),  clasmatocytes  (Ranvier), 
histiocytes  (Aschoff,  Kiyono),  and  macrophages  (Metchnikoff, 
Evans).  In  the  study  of  these  cells  endless  discussion  has 
arisen  on  their  relationship  to  other  cells  and  tissues  of  the 
body,  and,  on  the  relationships  which  exist  between  these 
cells  themselves,  as  they  were  known  to  have  certain  common 
characteristics,  chief  of  which  was  the  phagocytic  function. 
The  idea  of  classifying  these  diverse  elements  in  one  cell  group 
"the  Macrophage"  on  the  basis  of  this  phagocytic  function, 
as  suggested  by  H.  M.  Evans  (1915),  "The  Macrophages  of 
Mammals,"  has  been  adopted,  and  this  terminology  will  be 
used  to  designate  the  large  phagocytic  cells  that  appear  in  the 
circulating  blood  under  certain  conditions. 

These  macrophages  of  the  circulating  blood  have  been 
selected  as  they  are  particularly  adapted  for  the  study,  in 

4 

*  Received  for  publication  December,  1921. 


737116 


78  SIMPSON 

living  animals,  of  the  conditions  necessary  for  the  production 
of  macrophages  and  present  a  particularly  interesting  phase 
of  the  problem  of  cell  relationship,  due  to  their  possible  kin- 
ship with  the  normal  mononuclear  cells  of  the  blood. 

A  rapidly  increasing  number  of  reports  has  been  appearing 
in  the  literature  of  pathological  cases  in  which  large,  mononu- 
clear, phagocytic;  cells  were  found  in  the  circulating  blood. 
Several  pathological  conditions  have  been  represented  among 
the  cases  showing  this  response.  Mallory  (1898)  described 
large  phagocytic  cells  in  cases  of  typhoid  fever  and  ascribed 
to  these  cells  an  endothelial  origin.  MacCallum  (1903)  found 
that  the  mononuclear  cells  of  typhoid  were  poured  into  the 
circulation  from  the  thoracic  duct  so  rapidly  that  they  clogged 
many  of  the  small  branches  of  the  pulmonary  artery.  Ehrlich 
very  early  reported  the  presence  of  large  phagocytic  mono- 
nuclear  cells  in  cases  of  paroxysmal  haemoglobinuria.  Eason 
(1908),  also  Kommerer  and  Meyer  (1909),  have  described 
macrophages  characterized  by  phagocytized  red  blood  cells 
in  cases  of  paroxysmal  haemoglobinuria.  An  increase  in  large 
mononuclears  was  reported  in  the  blood  of  malaria  patients 
by  Pappenheim  (1900,  1901).  Bushnell  (1903)  and  Schilling 
(1919),  also  observed  these  unusual  cells  in  malaria.  Schilling 
has  reported  macrophages  in  several  other  conditions.  He  has 
made  his  most  careful  study  of  the  cells  as  they  appeared  in 
ulcerative  endocarditis  (1919).  The  results  of  this  study  will 
be  referred  to  again.  Leede  (1911)  and  Hynek  (cited  by 
Krezenecky  1911),  also  observed  these  cells  in  ulcerative 
endocarditis.  A  case  of  Libman's  subacute  bacterial  endo- 
carditis has  been  followed  in  connection  with  this  study  and 
the  cell  phenomena  observed  will  be  discussed  later  in  con- 
nection with  their  bearing  on  the  results  of  the  experimental 
work.  Kurpjuweit  (1903)  observed  mononuclears  larger  than 
normal  in  the  blood  of  a  case  of  carcinoma  ventriculi  —  no 
metastasis  in  the  bone  marrow.  The  cases  observed  by  Schil- 
ling other  than  tertiary  malaria  and  ulcerative  endocarditis, 
where  this  outpouring  of  large  mononuclear  cells  occurred, 
were  in  the  cases  of  monocyte  leukemia  (Reschad  and  Schilling 
1913),  in  latent  tuberculosis  (1919),  in  mesenteric  lymph  node 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD  79 

tuberculosis  (1919),  in  atypical  sepsis  (1919),  and  in  variola 
vera  (1916).  Some  of  the  other  workers  reporting  cells  of  this 
type  in  cases  of  infections  of  the  heart,  as  well  as  in  other  in- 
fections, in  cases  of  tumours,  and  in  cases  of  unknown  etiology, 
are  Fleischmann  (1915),  Hirschfeld  (1914),  Bingel  (1916), 
Rieux  (1911),  Kraus  (cited  by  Pappenheim  and  Fukushi 
(1913),  Netausek  (1916),  Kruzencky  (1917),  Kaenelson  (1919), 
Van  Nuys  (1907)  and  Bartlett  (1908). 

These  cells  were  first  produced  experimentally  by  Evans 
and  Winternitz  in  1911.  They  were  obtained  from  the  ear 
vein  of  rabbits  which  had  been  deeply  stained  from  chronic 
injections  of  Trypan  Blue.  The  first  report  of  the  experimental 
production  of  these  large  phagocytic  cells  in  the  circulating 
blood  was  given  by  Aschoff  and  Kiyono  (1913)  and  by  Kiyono 
(1913,  1914).  The  cells  were  found  in  the  circulation  after 
repeated  intravenous  injections  of  Lithium  Carmine.  Isamin- 
blau,  Trypanblau  and  Pyrrolblau,  Toluidinblau  were  found  to 
give  the  same  result  as  that  obtained  with  Carmine.  Collargol 
gave  similar  results.  Kiyono  believed  that  the  cells  produced, 
"  histiocy tes "  as  called  by  him,  were  a  normal  constituent  of 
the  blood  and  that  the  normal  quota  was  increased  under 
some  pathological  conditions  or  by  the  introduction  of  dye 
into  the  blood  stream. 

The  report  on  this  present  study  of  the  experimental  pro- 
duction of  macrophages  has  been  organized  as  follows: 

I.   Methods:  A.  Agents  used  to  produce  the  circulating  macrophages 
B.   Methods  of  study  of  the  circulating  macrophages. 

II.  Distribution  of  the  macrophages  in  the  blood  of  the  body  as  de- 
termined by:  A.   Seats  of  formation. 
B.  Filtration  of  the  large  cells  by  capillary  systems. 

III.  The  "Shower  Phenomena." 

IV.  Changes  in  the  blood  accompanying  Macrophage  Showers. 

V.  Relationship  between  the  macrophages  which  appear  in  the  cir- 
culation under  these  abnormal  conditions  and  the  normal  mono- 
nuclear  cells  of  the  blood. 


80  SIMPSON 

I.  METHODS 
A.  Agents  used  to  produce  the  circulating  macro phages 

The  following  materials  were  introduced  intravenously  into 
rabbits  with  the  hope  of  obtaining  the  circulating  macrophages : 

1.  Colloidal  dyes:  (a)  Niagara  Blue  26  (N.  A.  C.  Co.)  in  i  %  solution 

in  distilled  water.  The  stock  solution  was  filtered  and  boiled 
just  before  the  injection. 

(b)  Lithium  Carmine.  Prepared,  according  to  Kiyono  (1914).  5  % 
carmine  rubr.  opt.  (Griibler)  is  dissolved  in  a  solution  of  satu- 
rated lithium  carbonate  (saturated  in  the  cold).  The  solution 
was  filtered  just  before  the  injection. 

2.  More  coarsely  divided  material  held  hi  colloidal  state  by  protective 

colloids:  (a)  Red  gold.  The  protective  colloid  used  was  sodium 
lysalbinate.  The  dry  substance  contained  40  %  gold,  60  %  so- 
dium lysalbinate.  The  method  of  preparation  was  to  weigh 
doses  and  dissolve  in  sterile  distilled  water  just  before  use.  The 
particles  of  the  colloidal  gold  were  between  4  and  40  milli- 
micra  in  diameter. 

(b)  India  ink  (Higgin's) :  The  ink  was  injected  at  full  strength  or 
diluted  before  use  with  distilled  water  to  the  desired  concen- 
tration.   The  carbon  particles  were  just  at  the  limit  of  visi- 
bility (oil  immersion). 

(c)  Aqua  dag,  cone.  (Acheson  Oil  Dag  Co.) :  As  in  the  case  of  the 
india  ink  the  protective  colloid  was  unknown.   The  aqua  dag 
contained  a  high  concentration  of  ammonium  hydroxide.   The 
aqua  dag  was  weighed  and  diluted  hi  distilled  water  before  use. 
The  particles  were  just  visible  with  number  three  objective, 
ocular  No.  4. 

(d)  Lamp  black:  The  lamp  black  was  used  as  a  5  %  suspension  in 
i  %  gelatine  solution.   The  carbon  was  ground  for  one  half  hour 
in  the  form  of  a  paste  with  a  small  part  of  the  gelatine  solution 
before  the  rest  of  the  gelatine  solution  was  added.  100  to  200  c.c. 
portions  were  made,  and  were  sterilized  daily. 

3.  The  protective  colloids  of  group  2,  where  known,  were  injected  as 

controls,  (a)  Gelatine:  This  was  injected  as  in  the  case  of 
the  lamp  black  in  one  per  cent  solution.  As  before  the  solu- 
tion was  made  in  100  to  200  c.c.  portions  and  sterilized  daily. 
(b)  Sodium  lysalbinate:  Weighed  portions,  each  corresponding  to 
the  weight  of  sodium  lysalbinate  in  a  dose  of  the  colloidal  gold 
preparation,  were  dissolved  hi  sterile  distilled  water  just  before 
injection. 

Reasons  for  the  selection  of   these  agents  as  macrophage 
stimulants.  —  The  colloidal  dyes  of  Group  i  were  known  to 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD  8 1 

produce  the  response,  therefore,  were  used  as  a  reliable  method 
of  obtaining  the  cells  for  study.  Further,  the  colloidal  dyes 
were  to  furnish  the  smallest  colloidal  particles  in  a  series  of 
stimulating  agents  of  increasing  particle  size  (colloidal  dyes, 
colloidal  gold,  india  ink,  aqua  dag  and  lamp  black).  The  hope 
was  fostered  that  differences  in  phagocytosis  and  stimulation 
of  the  production  of  the  macrophages  might  be  found  to  run 
parallel  to  the  changes  in  particle  size,  i.e.,  this  class  of  cells 
might  show  greater  facility  in  handling  particles  of  certain 
sizes  and  there  was  a  chance  that  particles  of  some  sizes  would 
stimulate  the  mother  cells  to  proliferate  more  readily  than 
others.  All  were  supposedly  inert  substances  chemically,  so 
that  differences  observed  in  the  reaction  of  the  cells  could  be 
attributed  to  particle  size,  provided  the  protective  colloids 
were  not  themselves  active  stimulants.  Therefore  the  pro- 
tective substances,  Group  3,  had  to  be  tested  for  ability  to 
produce  macrophages.  When  it  was  found  that  these  protec- 
tive colloids  gave  the  same  typical  reaction  found  in  all  the 
substances  of  Groups  i  and  2  this  hope  of  testing  the  effect  of 
particle  size  was  lost.  The  experiment,  however,  did  show  that 
colloidal  substances,  protein  in  nature,  are  handled  by,  and 
cause  the  proliferation  of  the  mother  cell  of  the  macrophages. 

The  only  property  common  to  all  of  these  substances  in- 
jected was  the  presence,  in  each,  of  a  substance  in  the  colloidal 
state.  The  Niagara  Blue  2B  probably  represented  most  nearly 
a  single  colloidal  system,  though  the  Niagara  Blue  was  a 
commercial  product  and  probably  contained  some  crystalloid 
impurities.  The  Lithium  Carmine,  prepared  as  it  was  by  add- 
ing Carmine  to  a  saturated  lithium  carbonate  solution,  con- 
tained crystalloids.  The  other  substances  contained  at  least 
the  suspensoid  in  addition  to  the  colloid.  The  colloidal  gold 
and  lamp  black  preparations  were  probably  composed  of  just 
these  two  types  of  substances,  but  the  commercial  carbon 
preparations  contained  an  unknown  number  of  substances. 
The  aqua  dag,  judging  by  the  odor,  contained  a  high  concen- 
tration of  ammonium  hydroxide. 

The  injection  of  these  substances,  all  containing  colloidal 
particles,  may  have  presented  the  macrophage  mother  cells 


82  SIMPSON 

with  material  they  were  most  capable  of  handling  and  in  such 
unusual  quantities  as  to  cause  an  over  production  of  these 
cells.  The  part  played  by  the  coarse  particles  cannot  be  tested 
as  they  are  unstable  without  the  protective  colloids.  There  is 
still  the  possibility  that  the  crystalloids  stimulated  macro- 
phage  production.  This  is  certainly  the  next  step  necessary 
in  tracing  the  possible  significance  of  the  physical  state  of  the 
stimulating  agent  on  macrophage  production. 

Though  all  the  substances  selected  as  possible  macrophage 
stimulants  did  yield  the  macrophages  in  increased  numbers, 
however,  they  did  not  all  show  the  effect  at  the  same  rate. 
The  macrophages  were  first  seen  in  the  blood  at  rather  widely 
different  intervals.  The  blood  was  not  tested  for  the  presence 
of  the  macrophages  at  as  short  intervals  in  some  of  these  cases 
as  in  others,  also,  due  to  the  spasmodic  nature  of  the  macro- 
phage response  it  is  easy  to  overlook  the  macrophages  even 
when  the  animal  has  been  brought  to  the  point  where  the  cells 
appear  in  the  blood;  further,  the  macrophage  reaction  has  not 
been  studied  sufficiently  to  show  the  effect  on  macrophage 
production  of  the  interval  between  injections  and  other  such 
factors,  so  that  these  differences,  in  the  time  at  which  the 
macrophages  were  first  seen,  cannot  be  reliably  attributed  to 
differences  in  the  stimulant  action  of  various  substances  in- 
jected. The  data  on  the  time  and  dosage  at  which  the  reaction 
was  first  observed  have  been  collected  for  the  various  stimu- 
lants and  presented  in  the  following  table.  The  information 
is  at  least  interesting  on  the  basis  that  it  gives  an  estimate, 
perhaps  unnecessarily  large,  of  the  time  and  dosage  at  which 
the  reaction  can  be  obtained  in  these  substances. 

B.  Methods  of  study  of  the  circulating  macrophages 

i.  Methods  of  obtaining  the  blood. — During  treatment  the 
blood  was  obtained  from  the  ear  veins,  and  from  the  right  and 
left  ventricles.  At  autopsy  blood  was  obtained  from  both 
ventricles  and  from  the  large  internal  veins,  e.g.  iliac,  hepatic 
and  splenic.  The  blood  was  obtained,  if  possible,  from  animals 
under  ether  while  the  heart  was  still  beating.  In  some  cases 
blood  was  also  obtained,  at  autopsy,  from  hepatic  punctures. 


MACROPHAGES  IN   THE   CIRCULATING  BLOOD 


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84  SIMPSON 

The  peripheral  blood  was  soon  found  to  be  of  little  interest 
as  the  macrophages  do  not  reach  the  peripheral  blood  in  ap- 
preciable quantities  even  when  poured  into  the  right  heart  in 
enormous  numbers.  Therefore  heart  puncture  had  to  be 
resorted  to  as  the  only  method  in  the  living  animal  of  gaining 
an  adequate  idea  of  the  phenomena  occurring  within  the  cir- 
culating blood. 

The  criteria  used  to  determine  which  ventricle  had  been 
entered,  were  chiefly,  (i)  the  color  of  the  blood  and  (2)  the 
thickness  of  the  muscular  wall.  These  criteria  were  found  by 
repeated  experience  to  be  dependable.  The  first  criterion  is  of 
no  value,  however,  in  cases  of  recently  injected  Niagara  Blue 
.animals  as  the  blue  color  of  the  dye  obscures  the  natural  differ- 
ence in  color.  The  second  criterion  also  loses  its  value  when 
dense  adhesions  are  formed  binding  down  the  heart.  However, 
it  was  only  after  repeated  punctures  (50-100)  at  close  intervals 
that  adhesions  sometimes  interfered  seriously  with  this  test. 

2.  Methods  of  study. — Living  and  fixed  blood  was  used. 
The  living  blood  was  stained  supra-vitally  (Pappenheim,  see 
1907,  Rosin  and  Bibergeil,  1902,  1904)  with  Brilliant  Cresyl 
Blue  and  by  a  combination  of  Janus  Green  B  and  Neutral 
RedJ~The  fixed  smears  were  stained  with  combined  Jenner- 
Giemsa,  Wilson  stain,  oxydase  and  peroxydase  stains  e.g. 
Graham's  (1916-19)  oxydase,  Mcjunkin's  (1918),  oxydase, 
Mcjunkin's  (1920),  combined  benzidine-poly chrome  (Giemsa). 

Tissues  were  fixed  in  Kelly's  fixative  either  with  or  without 
injection  of  the  fixative  through  the  heart  and  were  imbedded 
in  paraffin  and  cut  into  sections  5  micra  thick.  They  were 
stained  by  Azur  II  and  eosin,  Delafield's  haemotoxylin  and 
eosin,  cochineal,  iron|haemotoxylin  and  by  Giemsa. 

.II.  DISTRIBUTION  OF  THE  MACROPHAGES  IN  THE 
CIRCULATING  BLOOD 

As  has  already  been  reported  by  Kiyono,  there  are  marked 
differences  in  the  distribution  of  macrophages  in  the  various 
regions  of  the  vascular  system.  The  blood  in  the  splenic  and 
hepatic  veins  is  very  rich  in  macrophages  during  the  produc- 
tion of  these  cells.  Kiyono  has  reported  that  of  these  two  veins 


MACROPHAGES  IN   THE  CIRCULATING  BLOOD  85 

the  hepatic  vein  always  contained  the  higher  per  cent  of  ma- 
crophages.  In  this  work  the  hepatic  vein  was  found  to  contain 
a  definitely  higher  per  cent  of  macrophages  in  the  cases  of 
animals  injected  with  gold,  sodium  lysalbinate,  gelatin,  and 
india  ink  (Nos.  2  and  3).  Kiyono  reported  that  the  inferior 
vena  cava,  right  ventricle  and  pulmonary  artery,  contained 
a  markedly  higher  proportion  of  these  cells  than  the  left  ven- 
tricle or  peripheral  circulation,  but  in  turn  contained  decidedly 
less  than  the  splenic  and  hepatic  veins.  The  mesenteric  and 
femoral  veins  contained  more  than  their  corresponding  arteries, 
but  the  numbers  present  were  small.  In  this  work  only  the 
right  and  left  ventricular  blood  and  that  from  the  splenic, 
hepatic,  femoral  and  peripheral  veins  was  analyzed.  The  order 
of  frequency  of  macrophage  occurrence  from  these  sources 
usually  was;  splenic  vein,  hepatic  vein,  femoral  vgin,  right 
ventricle,  left  ventricle,  and  peripheral  blood.  As  Kiyono 
states,  the  number  of  these  cells  in  the  peripheral  blood  is  very 
small,  even  after  chronic  dosage  and  in  conditions  where  the 
cells  are  being  poured  into  the  circulation  in  immense  num- 
bers (e.g.  constituting  90%  of  the  white  cells  present  in  the 
right  ventricle). 

There  are  probably  two  causes  for  this  unequal  distribution 
of  the  cells;  (A)  the  cells  have  originated  in  those  organs 
whose  venous  blood  contains  such  great  numbers  of  the  cells, 
(B)  the  cells  are  filtered  out  of  the  blood  as  the  blood  passes 
through  certain  capillary  beds,  chief  of  which  is  the  lung's 
capillary  system. 

A.  Origin  of  the  circulating  macrophages 

The  question  of  the  origin  of  the  macrophages  is  so  inti- 
mately associated  with  the  problem  of  relationship  between 
the  macrophages  and  the  normal  mononuclears  of  the  blood, 
that  the  main  discussion  of  this  question  and  the  contribution 
of  this  work  to  the  subject  will  be  given  with  consideration  of 
this  question  in  part  V.  Suffice  it  here,  to  say,  that  the  origin 
of  the  macrophage  that  had  been  suggested  by  the  high  count 
of  these  cells  in  certain  veins  has  been  substantiated  (Kiyono 
1913,  1914,  Evans,  H.  M.  1915,  Schilling  1919,  Mcjunkin 


86  SIMPSON 

1919,  etc.).    Kiyono's  evidence  on  the  sources  of  the  macro- 
phages  may  be  summed  up  as  follows: 

1.  The  sinusoids  or  capillaries,  and,  at  times,  the  tissues  of  the  organs 
drained  by  these  vessels  were  crowded  by  these  cells  (the  order  of 
importance  of  the  sources  decreasing  as  follows,  spleen  and  liver, 
bone  marrow,  lymphoid  tissue). 

2.  Mitotic  figures,  and  the  rounding  up  and  freeing  of  cells  marked 
by  the  accumulations  of  Carmine  were  observed  in  these  organs, 
(a)  In  the  spleen,  the  macrophages  arose  chiefly  from  rounding  up 

and  setting  free  of  reticulum  cells  of  the  red  pulp,  and  of  the 
lining  cells  of  the  venous  sinuses,  to  a  less  extent,  from  the 
reticulum  of  the  malphigian  bodies. 
(6)  In  the  liver  the  cells  arose  by  mitotis  of  the  Kupfer  cells. 

(c)  In  the  bone  marrow  they  arose  from  the  reticulo-endothelium, 
and  from  lining  cells  of  the  sinusoids. 

(d)  In  the  lymph  nodes  they  arose  from  the  reticulo-endothelium. 

The  reticulo-endothelium  and  the  capillary  or  sinusoid  en- 
dothelium  of  certain  organs  appear,  then,  to  be  the  active 
sources  of  macrophages.  H.  M.  Evans  (1915)  has  applied 
the  term  "  specific  endothelia"  to  the  endothelium  in  these 
tissues,  and  has  pointed  out  how  definitely  stimulation  of  the 
living  tissues  resulting  from  the  intravenous  injection  of  acid 
colloidal  dyes  is  confined  to  the  capillary  portion  of  the  vascu- 
lar endothelium  of  these  organs.  The  trunks  of  the  vessels  did 
not  respond  but  the  smallest  divisions  reacted  specifically. 
In  speaking  of  the  condition  in  haemolymph  nodes,  Evans 
said  "nothing  is  more  striking  than  the  abrupt  assumption  of 
brilliant  dye  granules  by  the  endothelium  of  a  venule  just  as 
it  enters  and  resolves  itself,  in  to  the  gland."  Kiyono  has  also 
noted  the  absence  of  the  Carmine  in  the  endothelium  of  the 
general  circulation.  Mcjunkin  gives  pictures  of  mitosis  in  the 
carbon-loaded  endothelial  lining  cells  of  capillaries  of  the  heart. 

That  the  stimulation  of  the  endothelium  of  the  five  organs 
represented  in  this  reaction  is  a  specific  response  due  to  a  par- 
ticular type  of  stimulating  agent  is  indicated  by  the  fact  that 
endothelium  in  other  parts  of  the  body  can  be  stimulated  by 
the  action  of  different  substances. 

The  proliferation  of  endothelium  in  other  parts  of  the  body 
is  well  known  to  occur,  e.g.  (i)  on  injury  of  endothelium  or  in 
embolism.  (Batchelor  and  Evans,  quoted  by  Evans  H.  M. 


MACROPHAGES  IN  THE  CIRCULATING  BLOOD  87 

1915,  injured  the  endothelium  of  larger  vessels  and  obtained 
proliferation.)  (2)  In  inflammatory  areas  the  endothelium  pro- 
liferates. (3)  The  endothelium  of  the  capillaries  of  the  lung 
is  supposed  to  assume  phagocytic  functions  and  to  proliferate 
forming  macrophages  on  intratracheal  introduction  of  Lithium 
Carmine  powder,  Permar  (1920-21). 

The  idea  is  involved  in  the  term,  "specific  endothelia," 
namely,  that  in  five  organs  of  the  body,  the  liver,  spleen,  bone 
marrow,  lymph  nodes  and  haemolymph  nodes,  the  endothelium 
is  differentiated  so  that  it  responds  almost  specifically  to 
colloidal  substances,  introduced  into  the  circulation.  The 
originator  of  the  term  " specific  endothelia"  felt  that  this 
reaction  of  the  endothelium  was  independent  of  the  chemical 
nature  of  the  stimulant  and  that  the  specificity  of  the  reaction 
was  due  to  the  physical  state  of  the  dye  which  was  injected. 
The  conception  that  the  reaction  was  independent  of  the 
chemical  composition  was  based  largely  on  the  variety  in 
chemical  constitution  of  the  dyes  producing  the  response  and 
on  the  supposedly  inert  chemical  nature  of  metallic  hydrosols 
producing  the  same  stimulation  (Evans  and  Schulemann,  1914, 
Evans,  Schulemann  and  Wilborn  1913). 

Difficulties  are  involved  in  testing  this  conception  with  the 
dyes.  If  dyes  with  small  particles  are  used  the  dyes  escape 
from  the  vascular  channels  because  of  their  increased  diffusi- 
bility.  In  these  cases  the  extravascular  macrophages  also  take 
part  in  handling  the  material  so  that  the  reaction  is  no  longer 
specific  for  the  macrophages  of  the  vascular  lining.  The 
attempt  was  made  in  this  work  to  test  the  conception  by  de- 
termining the  power  to  stimulate  macrophages  as  particle  size 
is  increased.  As  has  already  been  pointed  out  this  was  difficult, 
due  to  instability  of  unprotected,  colloidal  substances,  and 
due  to  the  ability  of  the  protective  colloids  themselves  to 
stimulate  endothelium.  The  effort,  however,  at  least  extended 
the  range  of  chemical  substances  giving  the  response,  and 
further  showed  stimulation  by  colloidal  particles  the  size 
found  in  gelatin  and  lysalbinate  solutions. 

B.  The  second  of  the  two  main  causes  for  the  great  differ- 
ences in  macrophage  distribution  in  different  parts  of  the 


88  SIMPSON 

circulation,  is  the  filtering  action  of  capillary  beds  traversed 
by  the  blood.  The  conception  of  a  filtering  action  by  the  lungs 
toward  large  cellular  elements  which  may  gain  entrance  to 
the  general  circulation  is  an  idea  introduced  by  Aschoff  and 
applied  by  his  pupil  Kiyono  to  the  explanation  of  the  low 
histiocyte  count  in  arterial  blood.  The  average  size  of  the 
macrophages  is  much  larger  than  the  polymorphonuclear  cell 
and  they  could  easily  be  caught  by  the  capillaries,  on  the  basis 
of  size  alone  regardless  of  other  peculiarities  which  might 
render  them  more  susceptible  to  be  caught  and  held  in  the 
capillaries.  Repeated  suggestions  are  found  in  the  literature 
that  cells  are  changed  after  assuming  phagocytic  functions  so 
that  they  are  caught  and  held  in  the  internal  organs.  The 
difference  in  the  macrophage  content  of  the  blood  on  the  two 
sides  of  the  heart  is  truly  spectacular  at  times.  The  right 
ventricle  has  been  observed  when  90%  of  white  cells  were 
macrophages  and  at  these  times  the  left  ventricle  would  not 
contain  enough  macrophages  to  show  in  a  usual  differential 
count  (i.e.  they  were  less  than  .1%  of  the  white  cells).  That 
capillaries  of  the  lungs  of  these  animals  were  packed  with  the 
large  phagocytic  cells  was  shown  by  the  autopsies.  It  is  diffi- 
cult to  understand  how  the  animals  remained  apparently 
normal  and  healthy,  when  kept  under  treatment  for  months 
at  a  time,  yielding  repeatedly  the  "  showers  of  macrophages." 
The  macrophage  production  was  not  continuous,  to  be  sure, 
but  the  periods  during  which  the  cells  were  poured  out  lasted 
from  a  few  hours  to  a  few  days  or  a  couple  of  weeks  and  this  is 
in  itself  difficult  to  understand,  considering  that  1000  to  9000 
macrophages  were  removed  from  each  cubic  millimeter  of 
blood  during  each  circulation. 

It  would  appear  that  either  the  degeneration  of  the  cells 
in  the  venous  blood  and  in  the  lung  is  very  rapid  or  that  the 
cells  migrate  rapidly  into  the  tissues  or  back  into  the  venous 
blood. 

The  study  of  the  tissues  showed  that  migration  of  the  cells 
was  at  least  a  part  of  the  process,  as  would  be  expected  from 
the  cases  of  anthracocis  and  from  Permar's  experimental 
studies  (1920-21).  The  peribronchial  lymph  nodes  contained 


MACROPHAGES  IN  THE  CIRCULATING  BLOOD  89 

far  more  of  the  phagocytic  cells  loaded  with  injected  materials 
than  the  lymph  nodes  elsewhere  in  the  body.  This  was  con- 
spicuous in  the  case  of  India  Ink  No.  3  which  had  been  under 
treatment  four  months.  The  macrophages  had  collected  in 
groups  and  were  scattered  irregularly  throughout  the  lymph 
node  (with  no  apparent  reason  for  the  position  of  the  cell 
clusters).  The  animal  had  been  treated  only  infrequently, 
every  week  or  two,  with  rather  weak  doses  (1/20)  for  the  last 
couple  of  months  of  its  life,  and  previous  to  the  time  of  its 
death  the  animal  had  not  received  a  dose  for  eleven  days,  so 
that  it  was  of  interest  to  find  at  autopsy  that  this  animal's 
lungs  were  practically  free  of  carbon  while  the  adjacent  lymph 
nodes  contained  carbon. 

Kiyono  suggests  that  the  glomeruli  of  the  kidney  may  also 
act  as  a  filter.  The  glomeruli  were  found  to  contain  phagocytic 
cells  almost  invariably,  though  in  small  numbers,  as  might  be 
expected  from  the  infrequency  of  the  cells  in  the  arterial  blood. 

III.   "THE  SHOWER  PHENOMENON" 

Description  of  the  typical  shower  of  macrophages.  —  During 
the  early  part  of  the  work  all  information  on  the  reaction  was 
obtained  from  the  peripheral  blood  and  from  autopsy  studies. 
By  these  methods  the  information  obtained  was  merely  of  a 
type  that  confirmed  previous  observations,  namely,  that  the 
content  of  macrophages  in  the  peripheral  blood  was  very  low 
even  after  prolonged  treatment,  and  that  the  splenic  and 
hepatic  veins  and  the  blood  of  the  right  ventricle  contained  a 
higher  macrophage  content.  R.  T.  Trotter  (1919)  had  used  the 
method  of  heart  puncture  to  some  extent  in  animals  treated 
intravenously  with  Niagara  Blue  2B.  In  some  cases  higher 
macrophage  counts  (9-1 1  %)  were  found  in  blood  from  the 
heart  than  in  peripheral  blood  taken  at  the  same  time.  Though 
it  was  difficult  to  tell  the  venous  from  arterial  blood  in  these 
animals  treated  with  Niagara  Blue,  however,  judging  from 
the  presence  of  the  macrophages,  the  blood  from  the  heart 
had  probably  been  obtained  from  the  right  ventricle.  The 
successful  application  of  the  heart  puncture  method  meant 


go  SIMPSON 

that  Kiyono's  results  on  the  distribution  of  macrophages  could 
be  confirmed  in  animals  while  still  living.  Further,  it  meant 
that  this  method  might  be  a  valuable  aid  in  obtaining  more 
information  on  the  nature  of  the  reaction.  So  the  heart  punc- 
ture was  tried  as  a  method  of  obtaining  blood  for  study,  and 
proved  to  be  so  satisfactory  that  this  was  adopted  as  the 
standard  method  of  studying  the  course  of  the  reaction  of 
living  animals  subjected  to  the  chronic  intravenous  injections. 
The  puncture  of  the  liver  might  also  be  a  valuable  method  of 
studying  the  macrophage  response  in  living  animals.  Judging 
from  some  results  of  liver  punctures  at  autopsy  and  from  the 
study  of  tissues  which  show  great  abundance  of  these  cells 
in  the  liver  before  they  are  poured  into  the  blood  in  appreci- 
able numbers,  the  method  might  give  information  about  the 
early  part  of  the  reaction  at  the  time  when  the  right  ventricle 
punctures  still  show  no  macrophage. 

The  vigorous  response  finally  obtained  in  these  chronically 
treated  animals  was  one  that  had  not  been  expected.  The 
possibility,  which  had  been  entertained,  that  the  macrophages 
of  the  right  ventricle  gradually  increased  as  the  condition 
became  more  chronic  had  to  be  discarded  as  soon  as  the  method 
of  heart  puncture  was  applied  to  the  problem.  When  an  animal 
was  treated  long  enough,  the  macrophage  reaction  would  ap- 
pear abruptly,  i.e.,  within  a  few  hours  after  a  given  injection 
an  animal,  which  previously  had  shown  no  macrophages, 
would  suddenly  give  differential  counts  in  which  17%  to  90% 
of  its  white  cells  from  the  blood  of  the  right  ventricle  were 

,  macrophages,  and  in  which  the  absolute  count  sometimes 
reached  9000  macrophages  per  cubic  millimeter.  After  a 
variable  duration  of  a  few  hours  to  a  few  days  the  reaction 
would  subside  as  quickly  or  more  quickly  than  it  had  begun, 
so  that  within  a  few  days  after  injection  the  animal  would 
again  show  no  macrophages  on  the  right  side  of  the  heart  (or 
at  least  too  few  to  appear  in  an  ordinary  differential  count). 

,  After  this  shower  of  macrophages  has  once  occurred,  another 
injection  would  usually  produce  a  repetition  of  the  shower. 

During  these  so-called  " showers  of  macrophages"  these 
cells  are  not  only  relatively  very  high,  but  their  actual  number 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD  91 

is  very  great  (1000-9000  per  mm.3)  indicating  an  immense 
rate  of  production  or  rate  of  outpour  from  the  haemopoietic 
organs  where  they  are  formed.  There  is  a  possibility  that  this 
large  number  of  macrophages  in  the  right  ventricle  does  not 
represent  the  number  poured  into  each  cubic  millimeter  of 
blood  during  each  heart  beat,  from  the  haemopoietic  organs, 
but  represents  an  accumulative  effect  due  to  the  backing  up 
of  the  macrophages  from  the  clogged  capillaries  of  the  lungs. 
The  pulmonary  valves  are,  however,  probably  adequate  to 
prevent  any  such  return  of  blood  into  the  heart.  So  the  count 
in  the  right  ventricle  would  be  a  measure  of  the  rate  at  which 
the  cells  were  produced  rather  than  an  indication  of  accumula- 
tion of  cells  produced  during  a  longer  time  interval.  The  sharp 
drop  of  macrophages  which  sometimes  occurred  (so  that  in 
less  than  thirty  minutes  they  completely  disappeared  from 
the  circulation),  also  indicated  an  immense  outpour  which 
could  be  suddenly  checked.  The  rise  and  fall  in  macrophage 
count  observed  in  the  right  ventricle  is,  then,  probably  a  true 
measure  of  the  rate  at  which  the  macrophages  are  being  poured 
out  of  the  internal  organs  during  the  shower  phenomenon. 

There  were  a  number  of  variations  in  the  shower  phenome- 
non, as  shown  in  the  animals  in  which  different  injection  ma- 
terials were  used.  The  time  elapsing  between  the  injection 
and  the  response  varied,  so  also  the  duration  of  the  response, 
and  the  time  required  to  reach  the  climax,  or  the  abruptness 
with  which  the  reaction  subsided,  were  all  subject  to  variation. 
Once  familiar  with  the  reaction  produced  by  a  given  stimulant, 
particularly  in  a  given  animal,  fairly  reliable  predictions  could 
be  made  on  the  course  which  would  be  expected  to  be  followed 
by  the  next  shower.  In  India  Ink  No.  3  for  instance,  the  suc- 
cessive showers  were  followed  for  a  period  of  about  two  and  a 
half  months.  In  this  animal  the  reaction  was  reasonably  sure 
to  follow  the  injection  in  about  two  days.  Another  day  or 
two  would  be  required  to  reach  the  climax  of  macrophage  pro- 
duction. In  other  cases  the  reaction  could  be  predicted  to 
occur  within  a  few  hours.  Thus  Niagara  Blue  2B  could  be 
counted  on  to  give  the  macrophage  shower  within  three  to 
seven  hours  after  the  injection. 


Q2  SIMPSON 

Some  of  the  data  obtained  have  been  selected  from  the  pro- 
tocols and  presented  in  the  following  short  table.  Very  little 
attempt  has  been  made  to  trace  the  causes  of  variations  in  the 
macrophage  reaction  or  to  evaluate  their  relative  importance, 
or  determine  their  relationship  to  one  another.  There  are 
probably  at  least  the  following  three  phases  to  the  problem 
however, 

1.  Differences  peculiar  to  the  substance  producing  the  cellu- 
lar response. 

2.  Methods  of  administration,  e.g.,  strength  of  solutions, 
frequency  of  dosage,  relation  of  injection  to  previous 
shower,  etc. 

3.  Peculiarities  of  the  individual  animals,  e.g.,  health,  age, 
sex,  etc. 

The  abrupt  disappearance  of  macrophages.  —  The  abrupt 
drop  in  macrophage  counts  shown  in  the  table  was  an  interest- 
ing variation  to  the  more  gradual  decrease  of  the  macrophage 
counts  which  from  the  earlier  work  with  India  Ink  No.  3  had 
come  to  be  regarded  as  the  usual  course  of  the  phenomenon. 
This  phase  of  the  shower  phenomenon  was  first  encountered 
in  an  attempt  to  confirm  a  high  macrophage  determination 
made  an  hour  or  two  earlier,  in  which  doubt  had  arisen  as  to 
the  possibility  of  contamination  of  the  blood  with  pericardial 
fluid.  The  second  heart  puncture  showed  an  entire  absence 
of  macrophages.  This  rinding  acted  to  strengthen  the  doubt 
in  the  mind  of  the  investigator  of  the  findings  in  the  first  case 
and  for  a  little  while  threw  into  question  the  previous  spectacu- 
lar macrophage  showers.  Subsequent  to  this  finding  several 
autopsies  of  animals  showing  high  macrophage  counts  in  the 
right  ventricle  were  made,  and  the  studies  of  the  right  ventric- 
ular blood  made  from  the  autopsy  board  failed  to  confirm  the 
high  macrophage  counts  which  had  been  obtained  from  the 
heart  puncture  studies  made  just  previous  to  death.  Cases 
occurred  in  which  88  %  of  the  white  cells  of  the  right  ventricle 
had  been  macrophages,  which  at  autopsy,  only  15-30  minutes 
later,  showed  almost  no  macrophages  (1.8%).  Still  assuming 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD 


93 


d 


Gradual, 
Abrupt, 
followed 


cS 
'O 
M    rj-  rj-  to  H 


O\  PO  ON 


I 

52 


a  d  g    . 

•§1^ 

O     O     <N     M 


I 


II 


H  O 

CN  CO 


§3 


o 


94  SIMPSON 

that  the  macrophages  had  been  present  but  that  they  had 
disappeared  suddenly,  efforts  were  made  to  eliminate  possible 
experimental  factors,  by  chance  responsible  for  the  disap- 
pearance. In  the  first  case  in  which  the  phenomenon  had  been 
observed  the  animal  had  been  placed  on  the  board  immediately 
before  the  puncture,  so  that,  in  this  case,  fright  was  a  possible 
cause  of  the  effect.  However,  the  same  result  was  obtained 
in  an  animal  that  had  been  on  the  board  during  the  two  hours 
intervening  between  the  first  and  second  punctures  in  which 
the  first  puncture  gave  a  high  macrophage  count  and  the  second 
no  macrophages.  The  use  of  the  ether  was  taken  into  consider- 
ation as  a  possible  cause  of  disappearance  of  the  macrophages. 
However,  animals  killed  by  a  blow  on  the  head  showed  the 
same  phenomenon.  It  was  only  late  in  the  work  that  an  animal 
was  obtained  at  autopsy  which  still  showed  the  macrophages 
in  the  right  ventricle  after  death.  Gelatin  No.  24  was  one  of 
these  cases.  The  count  in  Gelatin  No.  24  had  dropped;  however 
9.6  %  of  the  white  cells  were  still  macrophages.  On  the  autopsy 
of  Gold  No.  4  the  macrophage  count  in  blood  obtained  directly 
from  the  right  ventricle  was  even  higher  than  in  the  preceding 
heart  puncture  study.  Ether  had  been  used  in  both  cases. 
At  least  a  few  of  the  possible  factors  that  might  have  been 
involved  in  the  disappearance  of  the  macrophages  had  then 
been  eliminated.  The  death  of  an  animal  did  not  cause  a 
sudden  drop  necessarily;  ether  did  not  interfere;  also  fright 
at  being  placed  on  the  board  had  been  eliminated  as  a  cause 
of  the  sharp  drop  in  macrophage  count. 

It  was  fortunate  in  several  respects  that  an  autopsy  was 
obtained  in  which  the  macrophage  climax  was  still  at  its 
height  at  the  time  of  death.  Aside  from  the  fact  that  tissues 
were  obtained  while  the  animal  was  pouring  the  cells  into  the 
circulation  in  great  numbers  there  was  the  further  advantage 
that  the  possibility  had  been  definitely  removed  that  the  high 
macrophage  counts  obtained  at  the  heart  punctures  had  been 
the  result  of  contamination  with  pericardial  fluid.  The  criteria 
that  had  previously  been  employed  to  distinguish  pure  ventric- 
ular blood  from  blood  that  might  have  been  contaminated  by 
pericardial  fluid  were: 


MACROPHAGES  IN  THE  CIRCULATING  BLOOD  95 

(a)  The  blood  appeared  normal  and  was  shown  not  to  be  diluted  by: 

1.  No  decrease  in  viscosity,  —  as  measured  in  filling  the  diluting 
pipettes. 

2.  By  normal  red  blood  counts. 

3.  By  the  normal  total  and  differential  white  counts. 

(ft)  As  the  needle  pierces  the  ventricle  there  is  definite  sensation  of 
passing  through  dense  tissue.  This  criterion  is  probably  reliable 
except  in  cases  of  adhesions,  when  the  force  required  to  pierce  the 
fibrous  tissue  is  so  great  that  it  is  difficult  to  measure  the  small 
additional  force  required  to  enter  the  ventricle. 

(c)  The  macrophages  were  confined  to  the  right  ventricle.  If  the 
macrophages  had  been  obtained  from  the  pericardial  cavity  the 
bright  red  blood  from  the  left  ventricle  would  stand  an  equal 
chance  of  pollution.  These  showers  of  macrophages  were  never 
obtained  from  the  left  ventricle. 

These  criteria  were  quite  adequate  in  themselves  to  eliminate 
the  possibility  that  contamination  with  pericardial  fluid  was 
the  source  of  the  macrophages  and  the  sudden  disappearance 
of  macrophages  which  sometimes  was  shown  at  the  second 
heart  puncture  or  at  autopsy,  represented  a  new  phase  or 
peculiarity  of  the  macrophage  reaction,  which  had  not  pre- 
viously been  seen.  The  other  possible  contamination  is  equally 
improbable,  namely,  that  the  macrophages  were  obtained  from 
a  local  reaction  in  the  heart.  The  left  ventricle  was  pierced 
many  more  times  than  the  right  ventricle,  as  it  covers  such  a 
large  portion  of  the  ventral  surface  of  the  heart,  so  would  be 
far  more  subject  to  any  such  local  process.  Autopsies  showed 
that  the  endocardium  was  smooth  and  glistening  and  the 
valves  were  normal.  Section  of  the  musculature  showed  no 
focal  infection  resulting  from  the  punctures.  Local  reactions 
probably  did  not  contribute  to  the  macrophage  response,  and 
the  macrophage  counts  from  the  right  ventricle  are  probably 
reliable  measures  of  the  rate  of  outpour  of  these  cells  from 
the  liver  and  spleen.  The  only  explanation  that  can  be  offered 
for  this  abrupt  discontinuance  of  the  macrophage  reaction  is 
one  similar  to  that  discussed  by  Naegeli  (1919)  in  regard  to 
the  behavior  of  platelets.  After  intravenous  injection  of  pep- 
tone or  after  the  injection  of  many  colloidal  substances,  includ- 
ing gelatin,  the  platelets  disappear  from  the  blood  temporarily. 
The  claim  is,  that  the  platelets  are  not  destroyed  but  held  in 


96  SIMPSON 

the  liver  sinusoids.  The  presence  of  the  macrophages  in  the 
liver  sinusoids  after  the  macrophage  shower  has  ceased  might 
be  considered  to  furnish  further  proof  for  the  ability  of  certain 
internal  capillary  systems  to  exert  a  selective  action  on  the 
blood  passing  through  them. 

The  observation  of  the  macrophages  in  the  sinusoids  after 
the  sudden  disappearance  of  the  cells  from  the  peripheral 
blood  in  itself  throws  an  interesting  light  on  the  nature  of  the 
macrophage  reaction.  Apparently  the  number  of  the  cells  in 
the  circulating  blood  is  not  a  measure  of  the  rate  of  prolifera- 
tion of  the  mother  cells,  but  is  a  measure  of  the  rate  at  which 
the  cells  are  poured  out  from  the  haemopoietic  organs  in  which 
they  originate. 

Time  interval  between  "showers."  —  In  the  first  study  of 
the  " shower  phenomenon,"  in  India  Ink  No.  3  the  reaction 
was  allowed  to  subside  before  reinjection.  As  this  took  a  week 
or  two  there  was  a  considerable  interval  between  injections. 
When  in  cases  like  Gold  No.  4  difficulty  arose  in  obtaining  a 
repetition  of  the  phenomenon  after  the  condition  had  once 
been  established  in  the  animal,  the  suggestion  presented  itself 
that  the  rest  between  the  injections  had  been  an  essential 
factor  in  the  india  ink  animal  reactions,  and  that  the  failure 
in  these  cases  was  due  to  the  daily  dosage.  The  long  resting 
interval  followed  by  an  injection,  however,  did  not  prove  to 
be  essential  as  is  shown  by  the  last  two  climaxes  in  the  gold 
animal  where  the  second  reaction  was  produced  at  will  within 
twenty-four  hours  after  the  first  climax,  or  in  the  gelatin 
animal  where  the  reaction  could  regularly  be  obtained  within 
twenty-four  hours  of  the  injection. 

The  specificity  of  the  shower  phenomenon  to  the  agent 
producing  the  reaction.  —  The  possibility  that  the  macrophage 
reaction  was  specific  for  the  substance  which  had  produced 
the  reactive  condition  was  tested.  An  animal  was  brought 
to  the  point  of  macrophage  production  by  the  use  of  india  ink 
(India  Ink  No.  i)  and  was  then  injected  with  Niagara  Blue 
26.  A  reaction  followed  within  an  hour,  a  time  interval  more 


MACROPHAGES  IN  THE  CIRCULATING  BLOOD  97 

like  the  three  to  seven-hour  reaction  time  obtained  in  the 
Niagara  Blue  animals  than  the  twenty-four  to  forty-eight 
hour  preparatory  interval  observed  in  the  animals  treated 
with  india  ink.  The  reaction  is,  then,  probably  not  highly 
specific  for  the  causative  agent. 

A  clinical  illustration  of  the  " shower  phenomenon."  —  The 
blood  picture  of  a  case  of  Libman's  subacute  bacterial  endo- 
carditis was  followed  during  the  two  terminal  months  of  the 
course  of  the  disease.  C.  P.,  University  of  California  Hospital, 
1921.  The  case  occurred  in  the  medical  service  in  the  Uni- 
versity of  California  Hospital  and  the  author  is  indebted  to 
Drs.  Moffitt,  Kerr  and  Sampson  for  permission  to  observe  and 
for  aid  in  study.  The  patient  was  a  young  man  eighteen  years 
of  age.  The  illness  leading  to  the  patient's  death  (July,  1921), 
was  at  least  of  nine  months'  duration  (from  October,  1920). 
During  the  months  of  June  and  July  positive  blood  cultures 
were  obtained  of  non-haemolytic  streptococcus  viridans. 

From  the  time  of  entry  to  the  hospital  (May  5,  1920),  the 
case  showed  macrophages  (see  Figs.  31-37)  in  the  peripheral 
veins.  From  the  first,  the  number  of  macrophages  was  ob- 
served to  be  variable,  a  point  also  noticed  by  Schilling  (1919) 
in  his  careful  studies  of  ulcerative  endocarditis.  It  was  not, 
however,  until  June  n,  that  it  was  noticed  that  there  was  a 
parallel  here  to  the  shower  phenomenon  and  to  the  abrupt 
disappearance  of  the  macrophages,  which  had  been  such  a 
conspicuous  feature  in  the  experimentally  produced  reactions 
in  the  rabbit.  From  June  5,  1921  to  June  10,  1921,  daily 
studies  had  been  made  of  the  peripheral  blood.  The  macro- 
phage  count  had  varied  from  i%  to  1.5%.  On  June  n  the 
blood  from  the  ear  vein  was  examined  at  10  A.M.  and  found 
to  contain  14%  of  the  macrophages.  The  cells  were  of  such 
interest  and  the  blood  picture  so  conspicuous  that  the  blood 
was  taken  again  at  2  P.M.  The  blood  picture  had  returned  to 
the  usual  condition  and  there  were  but  i  %  of  the  macrophages 
present.  The  next  few  days  showed  several  marked  reactions 
(June  12,  16%,  June  14,  27%,  June  19,  8%).  On  June  23, 
the  phenomenon  of  the  sudden  disappearance  of  the  cells  was 


98  SIMPSON 

observed  again.  The  blood  at  12  M.  contained  48%  macro- 
phages,  at  4  P.M.  only  i%  of  these  cells  was  present.  The 
most  interesting  feature  of  this  case  had  been  the  high  counts 
of  macrophages  in  the  peripheral  blood.  Death  occurred  at 
a  time  when  the  number  of  the  cells  in  the  peripheral  blood  was 
very  low.  Blood  preparations  were  obtained  from  both  ven- 
tricles but  showed  no  differences.  (The  count  of  macrophages, 
both  sides  of  the  heart,  was  probably  below  i  %). 

The  possibility  of  determining  a  higher  macrophage  count 
in  the  right  ventricle  than  in  the  left  ventricle  was  therefore 
lost. 

Description  of  the  cells  produced  during  the  " shower."  — 
The  best  idea  of  the  morphology  and  staining  reactions  of 
these  cells  which  are  showered  into  the  circulation  can  prob- 
ably be  obtained  from  the  accompanying  drawings.  The 
characteristics  of  the  macrophages  can  be  summarized  as 
follows: 

A.  Morphology,     i.  Nucleus  —  size,  small  compared  to  the 
cytoplasm,  relatively  smaller  in  the  larger  cells. 

Shape,  round  oval  or  slightly  indented. 

Number,  usually  single,  though  large  cells  con- 
taining two  or  even  three  nuclei  are  not  un- 
common. 

Mitotic  figures  were  occasionally  observed  in 
circulating  cells. 

2.  Cytoplasm  —  contains  granules  and  vacuoles  in  vari- 
able quantities,  often  arranged  radially  about 
the  centriolar  apparatus. 

Contains  phagocytized  material,  whole  or  frag- 
mented red  and  white  cells  are  common. 

Contour — of  living  cell  — smooth  or  has  filiform 
pseudopodia,  also  bleb-like  pseudopodia  occur. 

Fixed  cells  —  smooth  contour  and  the  cell  is 
rounded  or  the  contour  may  be  irregular  (the 
spindle  shaped  cells  looking  like  endothelium). 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD  99 

B.  Staining,  i.  Giemsa-nucleus  —  rich  violet,  very  often 
vacuolar  in  structure  (Fig.  i).  Nucleoli,  a  cluster 
of  two  or  three,  pale  blue  staining. 
Cytoplasm,  packed  with  fine  azurophilic  gran- 
ules. Larger  purple  bodies  irregular  in  size  and 
number,  injested  cells  and  cell  fragments. 

2.  Oxydase  —  Graham's  (1916)  alpha  naphthol  pyronin 

technic.  F.  A.  Evan's  (1915,  1916)  alpha  naph- 
thol-par aphenylenediamine  technic . 
Mcjunkin's  (1920)  benzidine  polychrome  tech- 
nic (modification  of  Graham's  benzidine  technic 
(1919)  Mcjunkin's  (1920)  alpha  naphthol,  methyl 
violet  technic. 

All  methods  were  tried  and  were  found  to  be  nega- 
tive in  the  rabbit  macrophage,  except  for  evidently 
phagocytized  material  contained  in  the  cells,  e.g.,  in- 
cluded amphophils,  which  stained  brilliantly. 

3.  Supra- vital  stain.   Brilliant  Cresyl  Blue  (figs.  2,  9-15). 

Nucleus  —  unstained    at  the   low  concentrations 

used  for  the  study  of  the  living  cells. 
Cytoplasm 

Segregation  apparatus  —  violet,  variable  in 
amount,  usually  conspicuous  and  coarse  in 
structure  (phagocytized  red  and  white  cells  — 
stained  very  early). 

Mitochondria  —  unstained. 

Refractive  vacuoles  —  unstained. 

Non-refractive  vacuoles  —  almost  transparent, 

unstained. 
Neutral  Red  and  Janus  Green  B  (figs.  16-30,30-37). 

Nucleus  —  unstained. 

Cytoplasm 

Segregation  apparatus  and  phagocytised  ma- 
terial (red). 

Mitochondria  —  blue  green. 
Refractive  vacuoles — unstained  or  yellow  red. 
Non-refractive  vacuoles  —  unstained. 


100 


SIMPSON 


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IO2  SIMPSON 

C.  Functional  characteristics.  Phagocytosis.  It  seemed 
reasonable  at  the  outset  of  this  work  that  the  macrophages 
when  produced  would  be  marked  by  the  stimulating  substance 
(the  assumption  being  made  at  tfus  time  that  carbon  and  gold 
were  stimulants).  It  tyas  soon  discovered,  however,  that  the 
presence  of  the  stimulant  in  visible  quantities  was  the  excep- 
tion rather  than  the  rule  among  these  cells  poured  out  in  such 
great  numbers, 

In  a  few  cases  practically  all  of  the  immense  number  of  cells 
poured  out  did  contain  recognizable  amounts  of  injected  sub- 
stances (figs.  2,  8).  Similarly  heart  punctures  were  made  at 
some  times  when  far  more  fragments  of  phagocytised  red  and 
white  blood  cells  were  present  than  at  others.  Some  attempt 
was  made  to  find  out  what  relation  existed  between  the  extent 
of  phagocytosis  and  the  time  of  injection,  or  the  relation  to 
the  phase  of  the  reaction,  i.e.,  whether  the  cells  had  just  ap- 
peared in  the  blood  of  the  right  ventricle  or  were  decreasing 
in  number.  The  data  bearing  on  this  subject  have  been  col- 
lected in  the  following  table.  The  evidence  here  indicates 
that  in  animals  treated  with  India  Ink,  there  was  an  interval 
in  the  first  twenty-four  hours  after  injection  in  which  the 
macrophages  present  in  the  circulating  blood  contained  the 
maximum  number  of  phagocytised  particles  of  injected  ma- 
terial. After  forty-eight  hours  the  cells  poured  into  the  blood 
contained  very  little  carbon.  In  Niagara  Blue  animals  the 
maximum  content  of  phagocytised  material  was  observed 
between  three  and  seven  hours  after  the  injection.  The  fact 
that  the  cells  did  not  contain  the  injected  materials,  in  ap- 
preciable quantities,  during  the  greater  part  of  the  time  dur- 
ing which  they  were  poured  into  the  blood  stream  indicates  a 
great  overproduction  of  cells.  They  are  formed  in  such  num- 
bers that  they  are  still  sent  out  into  the  blood  stream  after 
the  stimulating  agent  was  no  longer  free  in  appreciable 
quantities  in  the  circulation. 

The  work  has  not  contributed  to  the  knowledge  of  the  con- 
ditions under  which  the  macrophages  acquired  their  load  of 
foreign  material,  i.e.,  as  to  possibility  of  phagocytosis  while 
circulating  in  the  blood.  Even  in  cases  where  macrophages 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD  103 

were  known  to  be  present  in  the  blood  at  the  time  of  injection, 
and  heavily  loaded  cells  resulted  from  the  injection,  there  was 
always  the  possibility  that  the  material  had  been  collected  by 
the  cells  while  still  fixed  or  while  present  in  the  slow  current 
of  the  sinusoids. 


IV.  CHANGES  IN  THE  BLOOD  ASSOCLA.TED  WITH  THE 
PRODUCTION  or  CIRCULATING  MACROPHAGES 

Many  changes  were  observed  in  the  blood  of  the  chronically 
injected  animals  other  than  the  appearance  of  the  macro- 
phages.  Most  of  these  changes  have  not  been  studied  sys- 
tematically enough  to  be  placed  on  a  strictly  quantitative 
basis,  however,  the  changes  were  often  striking  enough  to  be 
well  worth  noting.  The  following  variations  from  the  normal 
conditions  in  the  blood  were  observed : 

1.  Changes  in  viscosity  of  the  blood. 

2.  Changes  in  clotting  time. 

3.  Changes  in  platelet  counts. 

4a.  Changes  in  the  total  white  counts, 
b.  Changes  in  the  differential  white  counts. 

i .  Viscosity  changes  in  the  blood.  —  By  viscosity  is  meant 
the  internal  friction  of  a  liquid  as  measured  by  its  rate  of 
flow  through  a  small  tube  or  by  the  resistance  which  it  offers^ 
to  the  rotation  of  a  disc  in  it.  Great  changes  in  viscosity  were 
sometimes  observed,  when  handling  the  blood  from  the  right 
ventricles  of  animals  experiencing  the  macrophage  showers. 
The  dark  venous  blood  would  be  very  thick  and  stringy.  It 
would  be  extremely  easy  to  obtain  small  drops  in  making 
the  coverslip  preparations  and  particularly  easy  to  do  accu- 
rate work  in  filling  the  white  blood  pipette.  This  blood  con- 
trasted strongly  with  the  thin,  light  red  arterial  blood,  taken 
at  the  same  time  from  these  animals  from  the  left  ventricle. 
The  arterial  blood  came  from  the  hypodermic  needle  in  large 
drops  only  too  readily  and  would  rise  in  the  diluting  pipette 
very  quickly.  This  viscous  blood  was  only  obtained  from  the 
right  ventricle,  never  from  the  left.  It  was  found  only  during 


104  SIMPSON 

the  macrophage  showers,  but  did  not  invariably  accompany 
these  showers  (or  at  least  was  not  a  conspicuous  feature  during 
the  shower). 

It  was  observed  in  the  macrophage  showers  of  animals 
treated  with  india  ink,  gold  and  sodium  lysalbinate.  It  was 
not  observed  in  all  of  the  animals  in  which  showers  were  pro- 
duced by  these  substances  nor  in  all  the  showers  of  animals 
where  it  did  occur. 

The  phenomena  usually  associated  with  this  increase  of 
viscosity  were  (a)  the  formation  of  dense  flocculi  in  the  dilut- 
ing pipette  as  the  blood  was  mixed  with  i  %  acetic  acid,  (b)  the 
slow  clot  formation  or  no  clot,  (c)  the  low  platelet  counts. 

2.  Changes  in  clotting  of  the  blood  in  injected  animals.  — 
Profound  changes  were  sometimes  observed  in  the  clotting 
process  in  the  blood  of  injected  animals.  The  clotting  time 
might  merely  be  prolonged  and  a  typical  clot  would  finally 
be  formed  or  the  blood  might  not  clot  within  the  time  of  ob- 
servation (2  hours)  or  a  partial  clot  might  be  formed.  By  a 
partial  clot  is  meant  a  clot  which  involved  a  small  fraction  of 
the  blood,  or  the  whole  blood  sample  might  become  more 
viscous  or  semifluid,  and  still  remain  homogeneous.  The  latter 
process  might,  after  a  prolonged  interval  (30-40  min.)  result 
in  a  soft  clot  or  might  even  form  a  firm  clot  very  late.  These 
conditions  made  it  very  difficult  in  many  of  the  cases  to  decide 
on  what  should  be  called  the  coagulation  point. 

The  technic  used  in  making  the  clotting  time  determina- 
tions was  as  follows: 

The  blood  was  drawn  from  the  ventricle  into  a  i|  c.c.  paraffined  hypo- 
dermic. After  the  few  drops  necessary  for  making  the  supra-vitally 
stained  preparations  and  blood  counts  had  been  taken,  the  blood  from 
the  hypodermic  was  allowed  to  flow  down  the  side  of  a  paraffined  test 
tube,  taking  care  that  no  air  bubbles  were  introduced.  The  test  tubes 
were  i  cm.  in  diameter  and  were  filled  to  the  £  c.c.  mark.  The  blood  was 
then  quickly  covered  with  olive  oil  to  exclude  the  air.  The  whole  process 
from  the  time  the  blood  was  removed  from  the  heart  to  the  time  it  was 
covered  with  oil,  occupied  about  one-half  minute.  The  test  tube  was 
then  placed  in  a  convenient  receptacle  and  tipped  gently  from  time  to 
time  to  determine  the  time  at  which  the  blood  no  longer  flowed  freely 
but  remained  in  position  in  a  solid  mass  or  slipped  as  a  whole  down  the 
side  of  the  tube. 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD  105 

The  conditions  affecting  clotting  time  have  not  been  suffi- 
ciently studied  and  controlled  to  place  these  changes  in  clot- 
ting time,  which  accompanied  the  injections  on  a  good  quanti- 
tative basis.  The  clotting  time  was  often  observed  and  (see 
protocols)  found  to  vary  between  five  and  fifteen  minutes, 
and  though  these  may  have  been  significant  differences,  they 
have  not  been  taken  into  consideration  here  because  the  error 
in  technic  had  not  been  carefully  enough  determined  and 
controlled.  The  only  cases  which  are  regarded  as  of  interest 
were  those  in  which  the  type  of  clotting  had  been  markedly 
altered  or  those  in  which  a  typical  clot  was  not  observed 
within  a  reasonable  time,  for  instance,  one  to  two  hours. 

The  particularly  significant  thing  about  these  changes  in 
coagulation  was  the  fact  that  they  were  only  observed  in  the 
blood  taken  from  the  right  ventricle,  never  in  the  left  ventric- 
ular blood.  While  the  blood  of  the  left  ventricle  clotted  in 
the  normal  time,  blood  taken  from  the  right  ventricle  of  the 
same  animal,  practically  at  the  same  time  (or  one  to  two  hours 
before  or  after  the  blood  had  been  taken  from  the  other  ven- 
tricle) either  failed  to  clot,  or  only  clotted  partially,  or  clotted 
only  after  one  or  two  hours.  The  other  deviations  in  clotting 
were  also  observed  only  in  the  venous  blood. 

These  modifications  in  the  clotting  time  of  the  venous  blood 
almost  invariably  accompanied  the  crest  of  the  macrophage 
outpour,  so  that  it  was  the  blood  containing  these  unusual 
cells  which  had  these  serious  disturbances.  As  can  be  seen  in 
the  table,  however,  these  phenomena  were  observed  in  the 
venous  blood  of  the  injected  animal  in  a  few  cases  where  there 
were  no  macrophages  in  the  right  ventricular  blood.  This  was 
found  to  be  true  in  one  puncture  of  the  right  ventricle  of  the 
Gold  No.  4,  and  a  second  case  was  observed  in  the  venous 
blood  of  India  Ink  No.  2.  These  cases  might  be  explained  on 
the  basis  that  a  macrophage  shower  had  been  present  just 
before  examination  of  the  blood  but  the  macrophages  had 
undergone  an  abrupt  drop.  The  more  probable  explanation, 
however,  is  that  this  interference  in  clotting  was  the  effect  of 
the  injection.  Gold  No.  4  had  received  a  dose  of  colloidal  gold 
only  an  hour  before  the  study  and  the  India  Ink  animal  had 


io6 


SIMPSON 


received  an  unusually  high  dose  of  ink  (3^  c.c.  of  undiluted 
ink)  seven  hours  previous  the  heart  puncture.  The  inter- 
ference with  clotting  had  been  even  more  pronounced  a  few 

Relation  of  Delayed  or  Partial  Clotting  of  Right  Ventricular  Blood  to  its 
Macrophage  Content 


% 

Coagulatio 

a 

Substance 

Animal 

1921 

Time 

Macro- 
phages 

Right 
Ventricle 

Left 
Ventricle 

India  Ink  

2 

May  24 

1.2 

Partial  in  2  hrs. 

May  31 

9.31  A.M. 

•3 

No  clot  in  2  hrs. 

.... 

4.56  P.M. 

.O 

Clot  in  40  mins. 

June    3 

6. 

No  clot  in  2  hrs. 

June    4 

3.04  P.M. 

43-9 

No  clot  in  2  hrs. 

4.18  P.M. 

14  mm. 

June  1  6 

8.51  A.M. 

o 

Partial  in  2  hrs. 

India  Ink 

•2 

Mar.    7 

QO.O 

Mark'ly  slowed  * 

Mar.  12 

.... 

32.0 

Markedly  slowed 

Mar.  26 

87.7 

Markedly  slowed 

.... 

May    5 

1.35  P.M. 

QO.O 

No  clot  in  2  hrs. 

3.42  P.M. 



13  mm. 

Sodium 

22 

June  24 

1  2.  58  P.M. 

85.0 

Partial  in  2  hrs. 

Lysalbinate  .  .  . 

2.48  P.M. 

14  min. 

3.46  P.M. 

7.0 

Partial  in  2  hrs. 

6.57  P.M. 

O 

Clot  in  3  min. 

.... 

Gold  

4 

May  21 

10.21  A.M. 

o 

No  clot 

May  22 

12.45  P-M- 

35.5 

No  clot  in  \  hr. 

5-00  P.M. 

.... 

2-3  nun. 

May  23 

4.00  P.M. 

63.4 

No  clot  in  i  hr. 

Gelatin 

24 

May  24 

I.4.C  p.M. 

3!  min. 

1.57  P.M. 

2.0 

No  clot  in  2  hrs. 

NOTE:  The  macrophage  count  in  all  cases  was  zero  in  the  left  ventricle. 

*  In  some  of  the  earlier  macrophage  showers  in  which  attention  was  first  called  to  the  changes 
in  clotting  time  no  figures  were  obtained,  but  clotting  had  been  strikingly  enough  prolonged  to 
be  recorded  as  abnormal.  These  cases  have  been  included  in  the  table. 

hours  earlier  in  this  animal.  That  injections  of  the  materials 
used  could  interfere  with  the  coagulation,  quite  independently 
of  the  shower  phenomenon,  was  shown  by  injecting  10  c.c.  of 
undiluted  Ink  into  a  normal  animal  (India  Ink  No.  25) ;  within 


MACROPHAGES  IN  THE  CIRCULATING  BLOOD  107 

twenty  minutes  after  the  injection  grave  interference  in  the 
clotting  of  the  blood  was  observed.  See  discussion  by  Naegeli 
(1919),  and  the  work  of  Le  Sourd  &  Pagniez  (1906-13), 
Achard  &  Aynaud  1908,  Achard  &  Weil  (1907)  Sacerodotti 
(1893-1912).  Here  the  prolongation  of  clotting  time  after  the 
injection  of  colloids  (peptone,  gelatin,  etc.)  associated  with 
the  disappearance  of  platelets  has  been  reported. 

The  conclusion  is  not  justifiable,  however,  that  all  the  dis- 
turbances in  clotting  observed  in  these  experiments  were 
directly  caused  by  the  injections  and  were  not  related  to  the 
macrophage  showers.  The  parallel  between  abnormal  coagu- 
lation and  the  degree  of  the  macrophage  response  was  too 
clear  cut  for  such  a  conclusion. 

3.  Variations  of  the  platelet  count  in  chronically  injected 
animals.  —  In  counting  platelets  the  following  technic  was 
used:  Method  of  Ottenberg,  R.  &  Rosenthal,  N.  (1917).  A 
few  drops  of  blood  were  allowed  to  flow  from  the  hypodermic 
into  a  small  paraffine  cup,  immediately  after  the  blood  was 
obtained  from  the  heart.  This  blood  served  for  both  the  red 
cell,  white  cell  and  platelet  counts.  The  same  diluting  fluid 
(3%  sodium  citrate  containing  a  few  granules  of  Brilliant 
Cresyl  Blue)  was  used  in  making  both  red  cell  and  platelet 
counts.  The  preparation  was  shaken  two  minutes  and  allowed 
to  stand  ten  minutes  in  the  counting  chamber  before  the  count 
was  made. 

The  possible  significance  of  the  variations  in  platelet  counts 
between  150,000  and  200,000  has  not  been  taken  into  con- 
sideration here.  The  differences  in  platelet  counts  observed 
which  have  been  considered  were  those  outside  these  limits. 

The  outstanding  features  characteristic  of  the  platelet 
counts  as  can  be  seen  in  the  accompanying  table  entitled 
"Variations  in  platelet  counts  in  animals  receiving  chronic 
intravenous  injections"  were: 

(a)  enormous  reduction  or  disappearance  of  platelets  from 
the  venous  blood  obtained  from  the  right  heart,  while 
the  platelet  count  of  the  blood  of  the  left  ventricle  re- 
mained normal. 


io8 


SIMPSON 


(b)  the  decrease  in  platelets  in  the  blood  of  the  right  ven- 
tricle usually  ran  parallel  with  the  prolonged  clotting 
time  observed  in  this  blood  (therefore  also  parallel  with 
the  macrophage  showers). 

The  reduction  in  platelet  count  found  on  the  right  side  of 
the  heart  usually  ran  parallel  with  prolonged  or  abnormal 
clotting  time  in  this  blood,  as  they  did  with  the  macrophage 
showers.  However,  there  were  discrepancies  to  this  general 
rule  in  both  ways  possible,  i.e.  (i)  there  were  cases  in  which 
there  was  a  prolonged  clotting  time  while  the  platelets  were 
high  and  (2)  there  were  cases  where  there  was  a  low  platelet 
count  and  rapid  clotting. 

(i)  Cases  of  abnormal  clotting  when  the  same  blood  was 
rich  in  platelets. 


Animal 
No. 

Date 

Platelet 
Count 

Clotting 

Niagara  Blue  2B 

8 

May    2 

590,000 

Gelatinous  after  21  min.  and  re- 

mained in  this  condition 

May  1  5 

736,000 

Still  gelatinous  after  24  min. 

May  20 

364,000 

i%    hr.  fluid  —  gradually    thick- 

ened, no  definite  end  point 

June    9 

320,000 

45  min.  very  soft  dot  formed,  i\  hr. 

firm  clot. 

India  Ink  

•2 

May  17 

328,000 

14  min.  semifluid  mass  and  re- 

mained in  this  condition 

(2)  India  Ink  No.  i  on  June  23  is  a  case  in  which  the  clot- 
ting was  normal  while  the  number  of  platelets  was  very 
low. 

Though  there  was  a  parallel  in  most  of  the  cases  between 
disturbances  in  platelets  and  clotting,  there  was  not  complete 
agreement  in  the  occurrence  of  the  two  phenomena  as  might 
be  expected  from  the  complicated  nature  of  the  coagulation 
process.  There  were  some  hints  that  disturbances  did  occur 
in  the  other  factors  concerned  in  clotting.  In  several  cases 
where  there  was  a  delay  or  failure  to  clot,  and  a  decrease  or 
absence  of  platelets,  unusually  large  quantities  of  fibrin  were 
seen.  Fibrin  strands  are  very  inconspicuous  in  well-prepared, 


MACROPHAGES  IN  THE  CIRCULATING  BLOOD  1 09 

thin,  supra-vital  preparations  of  normal  blood.  If  seen  at  all 
they  occur  only  at  the  periphery  of  the  preparation  where  it 
is  thicker.  In  these  cases  the  fibrin  formed  throughout  the 
preparation.  Complicated  clumps  and  strands  of  fibrin  oc- 
curred. These  entangled  a  few  white  blood  cells  but  no  plate- 
lets or  very  few  platelets. 

The  fact  that  platelets  could  be  demonstrated  in  the  blood 
of  the  left  ventricle  even  when  completely  lacking  on  the  right 
side  of  the  heart  (as  shown  by  the  counting  chamber  and  the 
supra- vitally  stained  preparations)  indicates  (i)  that  the 
platelets  were  present  on  both  sides  of  the  heart  but  were 
more  fragile  in  venous  blood  than  in  the  arterial  blood  or  (2) 
that  the  platelets  are  absent  on  right  side  of  the  heart.  In 
this  case  their  presence  in  the  left  ventricle  would  have  to  be 
accounted  for.  It  would  be  possible  to  explain  platelet  for- 
mation as  due  to  fragmentation  of  macrophages  caught  in  the 
lungs.  There  was  some  evidence  in  the  work  that  explanation 
(i)  might  be  the  correct  explanation  of  the  low  counts  or 
absence  of  platelets  on  the  right  side  of  the  heart.  This  greater 
tendency  of  the  platelets  of  the  venous  blood  to  degenerate 
might  have  been  indicated;  (a)  by  the  hazy  outlines  and  the 
indefinite  clumps  formed  by  platelets  in  the  blood  of  the  right 
ventricle  as  seen  vin  supra- vital  studies  in  cases  where  low 
counts  were  reported  in  the  counting  chamber;  (b)  by  the 
presence  of  a  few  platelets  in  the  right  ventricular  blood  in 
supra-vitally  stained  preparations  where  none  were  distin- 
guishable in  the  counting  chamber;  (c)  by  the  presence  of  a 
purple  staining  precipitate  in  the  plasma  in  Giemsa  stains  of 
blood  from  the  right  ventricle,  in  cases  where  platelets  were 
missing  on  the  right  side  of  the  heart.  In  such  cases  the 
" precipitate"  was  not  found  in  the  stained  blood  of  the  left 
ventricle. 

Whether  one  assumes  that  the  platelets  are  absent  in  the 
venous  blood  or  whether  one  assumes  that  they  are  only  more 
fragile  in  this  blood,  both  explanations  involve  the  supposition 
that  a  remarkable  process  goes  on  in  the  lung,  whereby  the 
distinctly  abnormal  venous  blood  is  rendered  normal  by  the 
time  it  reaches  the  left  ventricle. 


1 10 


SIMPSON 


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MACROPHAGES  IN  THE   CIRCULATING  BLOOD 


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112  SIMPSON 

The  question  arises,  in  connection  with  the  inability  to 
demonstrate  platelets  in  venous  blood,  whether  the  disap- 
pearance of  the  platelets  was  due  to  the  injection  directly  or 
whether  the  phenomenon  was  associated  with  the  macrophage 
reaction.  The  work  of  LeSourd  and  Pagniez  (1906-13), 
Achard  and  Aynaud  (1908),  Achard  and  Weil  (1907)  and 
Sacerodotti  (1893-1912)  on  the  effect  of  injections  of  colloids, 
indicates  that  these  injections  can  have  pronounced  effects  on 
platelets.  There  are  very  many  instances,  however,  in  the 
records  of  this  study  in  which  the  platelets  did  not  decrease 
or  disappear  after  injections  in  cases  in  which  there  were  no 
macrophages  present,  whereas  most  venous  blood  which  con- 
tained macrophages  did  show  the  decrease  in  platelets.  There 
must  be  some  effect  on  platelets,  other  than  the  immediate 
effect  of  the  injection  connected  with  the  macrophages  reaction 
which  accounts  for  the  behavior  of  the  platelets. 

\/  4.  Variations  in  the  white  blood  counts.  —  (a)  Total  white 
counts.  Conspicuous  variations  in  the  total  white  count  were 
found  in  the  experimental  animals.  In  the  following  table 
cases  have  been  selected  in  which  total  counts  were  made  of 
the  white  cells  on  both  sides  of  the  heart  on  the  same  day. 
Some  of  these  cases  were  chosen  to  show  variations  in  the 
white  count  at  the  climax  of  the  macrophage  production. 
Other  cases  represent  the  periods  in  which  the  venous  blood 
contained  few  or  no  macrophages.  Other  counts  were  selected 
because  they  represented  the  condition  at  various  intervals 
after  injection. 
The  data  seem  to  demonstrate  at  least  the  following  points: 

1.  There  is  a  condition  of  leukopenia  in  the  blood  of  the 
right  ventricle  during  the  climax  which  is  not  shared  by  the 
blood  of  the  left  ventricle. 

2.  The  coaguli,  formed  by  the  action  of  the  acetic  acid  of 
the  diluting  fluid  immesh  the  macrophages  and  other  cells 
and  so  seriously  interfere  with  the  accurate  determination  of 
this  low  count  in  many  cases. 

3.  The  formation  of  the  heavy  coagulum  does  not  persist  long 
after  the  height  of  the  climax  of  the  macrophage  production, 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD 


Variations  in  the  Total  White  Counts  of  Injected  Animals 


Animal 

Date 

Time 

Injection 

Source 
blood 

Total  white 

Macro- 

phages 

India  Ink  No.  2  .  . 

May  10 

8  P.M. 

15  c.c. 

(1/40) 

8.45  P.M. 

R.V. 

marked  leuko- 

3 

penia  (differen- 

tial i  hr.  to 

count  64  cells) 

9-45  P-M. 

R.V. 

Leukopenia  no 

o 

longer  marked 

May  24 

R.V. 

5800  leukope- 

1.2 

nia  marked  (dif- 

ferential i  hr.  to 

count  100  cells) 

May  31 

8.45  P.M. 

.... 

R.V. 

13,000 

.6 

9.07  A.M. 

3%  c.c. 

undiluted 

9.31  A.M. 

R.V. 

28,600! 

•  3 

4.56  P.M. 

R.V. 

6,400 

.0 

India  Ink  No.  3  .  . 

May    5 

1.35  P.M. 

.... 

R.V. 

10,400  no  coag- 

90 

uli  mentioned 

3.42  P.M. 

L.V. 

25,000 

Lysalbinate 

June  24 

12.58  P.M. 

R.V. 

10,240  (count- 

85-1 

No.  22 

ing  all  coaguli 

in  ruled  field) 

2.48  P.M. 

.... 

L.V. 

13,900 

3.06  P.M. 

R.V. 

4,  700  no  coaguli 

40.1 

3.46  P.M. 

R.V. 

7,100  no  coaguli 

7.0 

6.57  P.M. 



R.V. 

1  7,  700  no  coaguli 

.0 

Gold  No.  4. 

May  21 

8.30  A.M. 

R.V. 

18,000 

o 

9.40  A.M. 

2O  C.C. 

IO.2I  A.M. 

.... 

R.V. 

15,000 

o 

9.OO  A.M. 

.... 

R.V. 

24,000 

o 

May  22 

12.45  P.M. 

.... 

R.V. 

failure  due  to 

35-5 

coaguli 

5-00  P.M. 

L.V. 

14,100 

May  23 

2.45  P.M. 

.... 

L.V. 

16,300 

4.OO  P.M. 

R.V. 

9,500 

63-4 

June  23 

10.30  P.M. 

R.V. 

13,440 

.6 

10.  1  1  P.M. 

L.V. 

14,600 

Gelatin  No.  24 

June  20 

counts  within  i  hr. 

R.V. 

2540!  (ihr.  to 

o 

of  each  other 

obtain  75  cells) 

L.V. 

7040 

0 

June  24 

1.45  P-M. 

L.V. 

9600 

R.V. 

3140 

June  25 

5.15  P.M. 

R.V. 

4600  (coaguli) 

61.0 

7.30  P.M. 

R.V. 

7000 

I.O 

114  SIMPSON 

so  ceases  to  interfere  with  the  demonstration  of  the  leukopenia 
before  the  end  of  the  shower. 

4.  Even  a  normal  white  count,  e.g.  8000  per  cu.  mm.,  in 
the  cases  where  the  macrophages  may  constitute  90%  of  the 
circulating  white  cells,  means  a  very  great  reduction  in  normal 
white  cells.   As  the  macrophages  disappear  from  the  blood  of 
the  right  ventricle  this  small  number  of  white  cells  in  the 
venous  blood  becomes  conspicuous  in  some  cases. 

5.  Recovery  from  the  leukopenia  is  rapid,  only  two  or  three 
hours  at  times. 

6.  This  period  of  leukopenia  may  be  present  in  these  in- 
jected animals  where  macrophage  showers  were  not  observed. 
This  is  amply  shown  in  the  following  table. 

7.  Although  the  leukopenia  could  often  be  detected  in  the 
counting  chamber  in  spite  of  the  formation  coagulum,  still 
it  was  a  point  of  great  satisfaction  to  be  able  to  confirm  the 
leukopenia  by  the  supra- vital  preparations.   The  supra- vitally 
stained  blood  gave  ample  evidence  for  the  leukopenia  in  the 
venous  blood, 

(a)  both  in  the  rabbits  where  there  were  no  macrophages 
(as  in  the  case  of  the  right  ventricular  blood  of  the  gela- 
tin animal). 

(b)  and  in  animals  where  macrophages  were  present  in  the 
venous  blood. 

In  both  these  types  of  cases  the  greatest  difficulty  was  found 
at  times  at  obtain  enough  cells  for  the  differential  count.  The 
preparation  was  examined  over  an  hour  in  some  cases  without 
seeing  a  hundred  cells,  while  the  differential  of  the  left  ventric- 
ular blood,  taken  from  the  animal  at  the  same  time,  could  be 
completed  in  a  few  miniutes. 

8.  The  leukopenia  is  often  succeeded  by  a  greater  number 
of  the  white  cells  than  the  normal  count.  The  increased  count 
is  common  to  the  blood  of  both  right  and  left  ventricle. 

(b)  Differential  white  blood  counts.  —  The  differential  blood 
counts  were  made  by  the  use  of  Giemsa  stain,  Wilson  stain, 
by  the  Graham  and  the  Mcjunkin  oxydase  technic,  by 


MACROPHAGES  IN  THE  CIRCULATING  BLOOD  115 

Mcjunkin's  benzidine-polychrome  stain,  and  by  the  supra- 
vital  stains  Brilliant  Cresyl  Blue  and  a  combination  of  Janus 
Green  B  and  Neutral  Red.  These  methods  were  not  of  equal 
merit  in  the  task  of  differentiating  the  mononuclear  cells,  a 
task  accomplished  best  by  the  supra-vital  method. 

Practically  all  the  differential  counts  were  made  by  Neutral 
Red  and  Janus  Green  B.  The  procedure  was  the  following: 
the  saturated  solutions  of  the  dyes  in  95  %  ethyl  alcohol  were 
mixed  just  before  use  in  the  proportions  Neutral  Red,  3  parts, 
to  Janus  Green  B,  4  parts.  The  thickness  of  the  smear  of  dye 
on  the  slide  can  be  easily  changed  as  desired  by  adding  a  few 
drops  of  95  %  alcohol  to  a  few  drops  of  the  dye  mixture  and 
by  the  rate  at  which  the  slide,  which  has  been  dipped  in  the 
dye,  is  drawn  over  the  surface  of  the  slide  on  which  the  dye 
is  to  be  spread.  Many  slides  can  be  prepared  in  this  way  at 
one  time  and  kept  ready  for  immediate  use  if  some  provision 
can  be  made  to  keep  the  slides  free  from  dust  and  lint.  The 
proper  sized  drop  of  blood  is  placed  on  the  cover  slip  and  the 
cover  slip  gently  dropped  on  the  stained  slide.  The  cover 
slip  is  then  quickly  rimmed  with  paraffin.  Provided  (i)  the 
glassware  is  clean,  and  (2)  it  has  an  even  surface,  and  (3)  the 
drop  is  of  a  proper  size,  chiefly  not  too  large,  so  that  the  prepa- 
ration is  not  too  thick,  and  (4)  the  dye  is  not  unevenly  streaked 
so  that  it  forms  ridges  which  prevent  even  spreading,  and  that 
(5)  a  very  small  time  elapses  between  the  removal  of  the  blood 
from  the  ventricle  and  the  completion  of  the  preparation,  the 
blood  will  spread  evenly  in  all  directions  to  the  margins  of 
the  coverslip.  In  a  good  preparation  there  should  be  only 
one  layer  of  red  blood  cells  which  are  non-crenated,  and  which 
are  evenly  spaced  throughout  the  preparation. 

The  concentration  of  stain  which  has  been  found  most 
satisfactory  for  counting  purposes  is  one  which  is  weak  enough 
to  leave  the  nucleus  unstained  for  at  least  15-30  minutes,  and 
yet  is  strong  enough  to  stain  the  specific  granules  and  segrega- 
tion apparatus  red  and  the  mitochondria  a  bright  green  blue. 
In  such  a  thin  lightly  stained  preparation  in  which  the  cells 
are  spread  out  between  the  slide  and  the  cover  slip  or  are 


Il6  SIMPSON 

creeping  over  the  glass  surface,  the  internal  structure  of  the 
cells  can  be  studied  to  good  advantage. 

In  identifying  the  granular  cells,  the  greater  size,  oval 
shape,  and  refractive  appearance  of  the  eosinophilic  granules 
distinguish  these  cells  from  the  amphophils.  The  basophil 
granules  stain  a  far  deeper  red  with  the  Neutral  Red  than  the 
amphophil  or  eosinophil  granules,  and  the  granules  are  small 
and  even  in  size.  Great  similarity  may  exist  between  the  size 
and  staining  reaction  of  the  mast  cell  granules  of  the  basophils 
and  the  segregation  apparatus  of  the  monocytes,  so  that 
what  difficulty  there  is  in  differentiating  the  basophil  comes 
about  in  its  distinction  from  monocytes,  rather  than  from 
difficulty  in  separating  it  from  eosinophile  or  amphophile  cells. 
The  greater  mitochondrial  content  of  the  monocyte  when  com- 
pared with  that  of  the  basophil  renders  the  Neutral  Red- 
Janus  Green  mixture  superior  to  the  Brilliant  Cresyl  Blue 
supra- vital  staining  in  differentiating  basophils.  The  Janus 
Green  stains  the  mitochondria,  the  Neutral  Red  stains  the 
segregation  apparatus,  while  only  the  latter  structures  are 
stained  with  Brilliant  Cresyl  Blue.  It  is  true  that  as  a  rule 
the  mast  cell  granules  show  a  more  marked  metachromasia 
with  Brilliant  Cresyl  Blue  than  the  segregation  apparatus  of 
the  monocytes  but  this  is  not  so  important  in  differentiation 
as  the  mitochondrial  content. 

In  classifying  the  mononuclear  cells,  they  were  divided  into 
lymphocytes  and  monocytes.  Briefly  summarized,  the  char- 
acteristics used  to  recognize  the  two  groups  of  mononuclear 
cells  are: 

Lymphocytes:  size,  less  than  the  size  of  a  red  blood  cor- 
puscle to  cells  greater  than  polymorphs,  the  so-called 
large  lymphocytes. 

nucleus,  usually  single,  sometimes  indented. 

cytoplasm,  apt  to  be  small  in  proportion  to  the  nucleus, 
glassy. 

segregation  apparatus,  granules 

(1)  1-8  the  average  would  be  (3-4  granules). 

(2)  mitochondria  in  great  numbers  (40  or  60  or  greater). 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD  1 17 

Monocytes:  size,  as  large  as  polymorphonuclear  cells  or 
larger. 

nucleus,  usually  indented,  horse-shoe  in  shape  and  more 
active. 

cytoplasm,  ground-glass  appearance,  suggesting  innumer- 
able granules. 

granules,  segregation  apparatus,  numerous  granules  (40- 
60). 

mitochondria,  equal  in  number  to  segregation  apparatus. 

The  contrast  between  the  glassy  cytoplasm  and  the  small 
number  of  granules  in  the  segregation  apparatus  of  the  lympho- 
cyte, the  more  opaque  cytoplasm  and  numerous  granules  of 
the  segregation  apparatus  in  the  transitionals,  furnish  the 
important  points  of  differentiation  between  the  two  cell  types. 
These  points  of  difference  have  made  the  supra-vital  method 
more  reliable  than  Giemsa  or  the  oxydase  stains  in  distinguish- 
ing the  monocytes  and  lymphocytes  of  the  rabbit.  With  the 
supra-vital  stain  the  separation  between  these  two  types  of 
cells  has  proven  to  be  as  sharp  in  the  rabbit  as  it  had  previously 
been  found  in  human  blood. 

The  presence  in  lymphocytes,  which  are  stained  by  Janus 
Green  and  Neutral  Red  of  numerous  blue  green  granules  and 
only  a  few  bright  red  ones,  while  the  red  structures  are 
abundant  in  the  monocytes,  makes  the  lymphocytes  easier  to 
differentiate  from  the  monocytes  than  the  single  segregation 
apparatus  stain  of  a  dye  like  Brilliant  Cresyl  Blue. 

The  rate  at  which  living  preparations  begin  to  show  signs 
of  degeneration  varies  greatly  with  the  temperature.  In  this 
work  it  was  found  that  in  cool  weather  the  preparations  could 
be  used  for  two  hours  or  more.  There  is  always  time  for  a 
reliable  differential  count  of  several  hundred  cells.  As  a  policy 
to  be  recommended  in  using  the  supra-vital  method,  it  is  best 
to  use  the  preparation  at  once,  in  order  to  obtain  the  most 
clear  cut  differentiation  of  cell  structures. 

The  normal  white  cell  counts  of  rabbits  have  been  given 
various  values,  as  can  be  seen  in  the  table  on  page  118. 


u8 


SIMPSON 

Normal  Diferential  White  Counts  of  the  Rabbit 


Author 

Totals 

Mononuclear  Group 

Am- 
phophils 

Eosin- 
ophils 

Baso- 
phils 

Lym- 
pho- 
cytes 

% 
Large 
mononu- 
clears 

Transi- 
tionals 

Kanthack  and 
Hardy  

70-80 

2-6 

20-30 

1-2 

2.5 

Proscher  

60-65 

.... 

33-40 

Q-.57 

4-8 

Tallquist  and 
Willebrand  

.... 

20-25 

2O-25 

.... 

45-55 

•5--3 

2-5 

Lindberg  

36 

7 

45-5 

i-5 

6 

Lowit 

31-9 

3 

60.4 

o-.8 

1.6-3 

Brinkerhoff  and 
Tyzzer 

12,000-4,000 

45-55 

2-8 

40-50 

•5-i 

4-8 

Moss   

12,800-6,520 
14,300-5,320 
13,000-5,900 

37-68 
12-76 
5-85 

4-17 
2.5-13-5 
4-14.5 

22-46 
12-76 
22-42 

Q--35 
o-.i5 

•5-8.5 

2-6 
5-4 
3-8 

Mcjunkin  (basis 
phagocytosis  of 
carbon)      

1.1—4.7 

Evans,  F.  A. 
(basis  oxydase 
reaction)  

5- 

Basis  supra- 
vital  stain  



approxi- 
mately 
3-5 

The  great  variations  in  the  percentage  and  total  counts  of 
the  white  cell  of  the  rabbit  is  evident  especially  in  the  results 
of  Moss  (1911)  who  studied  the  same  animal  for  representative 
periods.  The  animals  were  kept  under  practically  the  same 
conditions  as  to  sanitation,  feeding,  etc.,  and  even  when  the 
daily  examination  was  made  at  the  same  time  of  the  day,  the 
variation  occurring  could  amount  to  100%.  The  same  animal's 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD  119 

count  varied  greatly  both  at  different  times  of  the  same  day 
and  on  successive  days.  These  variations  must  be  kept  in 
mind  when  judgments  are  made  on  the  significance  of  changes 
in  white  cells  after  experimental  procedures. 

The  most  outstanding  feature  of  the  behavior  of  the  white 
blood  cells  in  the  chronically  injected  animals  was  the  over- 
production of  normal  blood  cells  after  the  macrophage  shower 
had  subsided,  due  largely  to  an  amphophil  increase. 

On  the  whole,  the  results  did  not  give  clear  evidence  that 
the  changes  in  the  lymphocytes  and  amphophils  were  related 
definitely  to  the  mechanism  involved  in  the  outpouring  of 
macrophages,  or  that  these  changes  were  related  to  the  in- 
jection independently  of  the  macrophage  response. 

The  changes  in  the  monocytes  during  injection  and  during 
the  macrophage  showers  are  so  intimately  related  to  the  prob- 
lem of  the  relationships  between  the  monocytes,  of  normal 
blood  and  macrophages  that  this  matter  will  be  discussed  in 
Part  V. 


V.  THE  RELATIONSHIP  OF  THE  MACROPHAGES  TO  THE 

MONONUCLEAR  CELLS   OF   THE  BLOOD 

Within  the  last  few  years  great  interest  has  been  attached 
to  the  possibility  of  our  being  able  to  recognize  two  cell  strains 
among  the  normal  mononuclear  blood  cells.  Evidence  has 
accumulated  to  sustain  the  view  that  the  so-called  transitionals 
and  mononuclears  of  Ehrlich  are  not  related  to  the  lympho- 
cytes but  constitute  a  third  major  cell  class.  The  term  mono- 
cyte  has  been  employed  in  this  paper  for  this  cell  group  to 
which  so  many  other  terms  have  been  applied. 

With  the  development  and  modification  of  the  Romanowsky 
method  for  staining  fixed  blood  preparations  it  has  come  to  be 
recognized  that  a  group  of  the  mononuclear  ceDs  was  char- 
acterized by  the  possession  of  an  abundant,  fine,  dust-like, 
azurophilic  granulation.  This  type  of  granulation  distin- 
guishes them  from  lymphocyte  characterized  by  a  different, 
scantier  azurophilic  granulation  discovered  by  Michaelis  and 
Wolff.  These  differences  have  been  best  expressed  by  Naegeli 


120  SIMPSON 

(1919)  who  calls  attention  to  the  fact  that  the  lymphocytic 
granules  only  occur  in  a  portion  of  the  lymphocytic  cells, 
that  they  are  brilliant  red,  scanty  in  number  and  of  irregular 
size,  some  of  them  being  coarse. 

A  second  method  which  in  some  instances  would  appear  to 
separate  clearly  these  two  cell  types  is  the  oxydase  method. 
This  reaction  is  usually  given  by  monocytes  and  myeloid  cells, 
but  not  by  the  lymphocytes. 

A  variety  of  workers  have  been  concerned  here  of  whom 
Brandenberg,  Meyer,  Winkler,  W.  H.  Schultze,  and  Gierke, 
may  be  mentioned.  Schultze,  Dunn  and  Naegeli  as  a  result  of 
extended  experience  with  the  method  are  convinced  that  under 
no  circumstances  is  the  reaction  given  by  lymphocytes. 

Mcjunkin  has  proposed  the  so-called  phagocytic  test  for  the 
recognition  of  the  monocyte  cell  to  which  he  applies  Mallory's 
term  "endothelial  leucocyte."  These  cells  phagocytise  carbon 
particles  whereas  lymphocytes  do  not. 

Against  the  Giemsa  method  for  differentiating  these  cells  is 
to  be  urged  the  difficulty  of  its  application  in  some  animal 
forms.  The  experimental  animal  used  here,  and  the  one  most 
available  for  intravenous  procedures  —  the  rabbit  —  is  a  case 
in  point.  Here  the  basophilia  of  the  monocytic  cytoplasm 
obscures  or  prevents  the  demonstration  of  the  azurophilic 
granules. 

The  oxydase  method  would  especially  appear  to  be  unrelia- 
able.  Indeed  even  its  adequacy  for  distinguishing  myeloid 
cells  has  been  questioned  (Rosen thai).  It  is  significant  that 
some  of  the  most  prominent  students  of  the  problem  of  the 
monocytes  deny  that  the  reaction  is  given  by  these  cells 
(Schilling,  Schlenner).  Against  the  Mcjunkin  phagocytic 
method  for  the  recognition  of  monocytes  is  to  be  urged  that  it 
is  difficult  or  impossible  to  secure  conditions  forcing  the  ex- 
hibition of  these  behaviors  on  the  part  of  every  monocyte  cell. 

On  the  other  hand  vital  staining  of  the  blood  constitutes  an 
adequate  method  for  the  recognition  of  these  cells,  as  has  been 
brought  out  in  a  previous  paper  (Simpson,  M.  E.,  1921).  The 
method  is  as  useful  in  the  study  of  rabbit  as  it  was  found 
to  be  in  the  study  of  human  blood.  In  fact  it  has  been  the 


MACROPHAGES  IN  THE  CIRCULATING  BLOOD  121 

only  method  found  reliable  for  the  recognition  of  the  mono- 
cytes  in  rabbits'  blood. 

One  of  the  central  aims  of  the  present  study  has  been  the 
detection  of  the  relation  of  the  great  phagocytic  cells  here 
produced  —  the  macrophages  —  to  the  blood  mononuclear. 
As  is  well  known,  Goldmann,  Aschoff,  Kiyono  and  Evans  have 
denied  any  relation  and  regarded  the  macrophages  as  stranger 
cells  of  tissue  origin,  hence  Aschoff's  term  "histocyte." 

The  criteria  for  the  separation  of  two  groups  of  mononuclear 
blood  cells  which  have  just  been  rehearsed  have  been  applied 
to  blood  containing  the  giant  phagocytes  or  macrophage  cells. 

All  of  these  methods  show  a  striking  parallelism  between 
the  macrophages  and  monocytes,  a  parallelism  not  shown  by 
the  lymphatic  cells.  One  is  called  upon  to  decide  whether 
there  is  merely  an  affinity  or  an  identity  in  the  case  of  these 
two  groups  of  cells.  The  identity  of  macrophages  and  mono- 
cytes or,  at  any  rate,  the  view  that  both  represent  a  common 
cell  strain  has  been  emphatically  championed  by  Mallory, 
Schilling,  Mcjunkin,  and  Schlenner.  The  chief  arguments  in 
favor  of  such  a  view  are:  (i)  The  existence  of  all  intermediate 
morphological  cell  types  (figs.  16-20).  This  has  been  brought 
out  prominently  by  Victor  Schilling  and  was  a  fact  sub- 
stantiated by  the  present  work  during  the  phenomenon  of 
macrophage  showers.  The  vital  method  shows  it  just  as  con- 
clusively as  Schilling's  application  of  Giemsa  to  human  ma- 
terial. (2)  The  overproduction  of  both  cell  types  occurs 
simultaneously  in  some  clinical  and  experimental  conditions. 
Reschad  and  Schilling  (1913)  deserve  great  credit  for  recogniz- 
ing clinical  conditions  in  which  occur  overproduction  of  the 
monocytes  (monocytoses  and  monocyte  leukemias).  In  such 
instances  the  blood  picture  shows  a  pure  monocytosis.  Schil- 
ling has  discovered  typical  macrophage  production  in  the 
liver  and  spleen.  He  consequently  feels  that  such  cases  con- 
stitute a  satisfactory  link  in  the  chain  of  cases  starting  with 
pure  monocytoses  to  outspoken  macrophagocytoses,  where  the 
giant  cells  are  frequent  in  the  blood.  For  several  years  F.  B. 
Mallory  has  called  attention  to  the  proliferation  of  endothelium 
and  consequent  formation  of  macrophages  in  the  internal 


122 


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MACROPHAGES  IN  THE   CIRCULATING  BLOOD 


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124  SIMPSON 

organs  in  many  diseases.  He  has  not  hesitated  to  identify 
these  elements  with  the  monocytes  of  normal  blood  and  has 
applied  the  term  endothelial  leucocyte  indifferently  to  all  of 
these  cells.  Mcjunkin  has  sought  to  establish  this  view  at 
one  time  by  demonstrating  that  the  liver  endothelium  and 
the  monocytes  showed  the  oxydase  reaction  in  common  and 
at  another  tune  by  demonstrating  that  both  cells  engulfed 
carbon  particles  administered  intravenously.  Moreover,  Mc- 
junkin has  employed  the  oxydase  test  to  recognize  monocytes 
in  the  guinea  pig,  where  he  has  reported  that  a  variety  of 
procedures  gives  an  overproduction  of  these  cells  —  a  mono- 
cytosis. 

There  is  some  evidence  for  the  view  that  the  cell  types  em- 
braced by  the  terms  macrophage  and  monocyte  are  separate, 
no  less  than  are  lymphocytes  or  monocytes.  For  in  spite  of 
the  striking  morphological  resemblances,  for  instance,  be- 
tween small  macrophages  and  monocyte  cells,  differences 
occur.  These  differences  may  be  discussed  as  follows: 

i.  Macrophages  stain  vitally;   monocytes  do  not  do  so. 

Evans  (1913-15)  has  shown  that  the  macrophages  are  well 
characterized  by  their  ability  to  receive  and  segregate  colloidal 
substances  comparable  to  the  benzidine  dyes  which  are  taken 
up  electively  by  them.  The  monocytes  do  not  do  this.  In 
cases  here  reported  of  chronic  dosage  of  rabbits  with  Niagara 
Blue,  although  there  was  invariably  an  increase  in  the  per- 
centage of  monocytes  in  the  blood  (which  in  some  cases 
amounted  to  more  than  30%  of  the  total  white  count),  under 
no  condition  did  the  monocytes  phagocytise  the  abundant 
vital  dye.  This  experience  emphasizes  and  corroborates 
similar  reports  by  Evans,  employing  benzidine  dyes,  and  by 
Aschoff  and  Kiyono,  employing  Lithium  Carmine.  Downey 
contends  that  this  difference  in  vital  stainability  is  due  to  the 
fact  that  the  monocytes  are  in  the  circulation,  which  in  some 
obscure  way  inhibits  dye  phagocytosis,  whereas  the  macro- 
phages are  wedged  in  certain  capillary  beds.  But  this  con- 
tention is  negated  by  an  instance  in  which,  having  found  a 
high  proportion  of  circulating  macrophages,  a  fresh  dose  of 
Niagara  Blue  could  be  seen  to  enter  at  least  70%  of  these  cells. 


MACROPHAGES  IN  THE  CIRCULATING  BLOOD  12$ 

2.  Monocytes  and  macrophages  do  not  always  occur  to- 
gether in  the  fluids  or  organs  of  the  body. 

Some  years  ago  Patella  (1909)  and  Crescenti  (1904)  in- 
dependently reported  the  absence  of  monocytes  from  the 
thoracic  duct  lymph.  Their  work  could  be  open  to  the  objec- 
tion that  these  observers  possessed  no  reliable  criteria  for  the 
separation  of  this  cell  group  from  lymphocytes.  Recently 
Lejeune  (1914-15)  employed  Giemsa's  stain  and  established 
the  absence  of  monocytes  from  the  thoracic  lymph.  Nor  with 
the  method  of  supra-vital  stains  have  monocytes  been  seen 
by  us  in  taps  of  the  duct.  On  the  other  hand,  the  lymph  in 
animals  stained  with  Niagara  Blue  always  carries  a  fair  num- 
ber of  unmistakable  macrophages.  This  is  easy  of  explana- 
tion, for  the  lymphatic  glands  are  well  known  seats  of  the 
endothelial  proliferation  which  forms  macrophage  cells  and 
the  latter  throng  both  peripheral  and  medullary  lymph 
sinuses  (Evans,  Kiyono).  It  would  appear  that  we  cannot 
have  in  the  lymphatic  gland  a  source  of  monocyte  production 
and  yet  this  is  nevertheless  one  of  the  most  active  sources 
for  macrophage  production. 

Some  attempt  was  made  to  analyze  the  blood  from  the  possible  sites 
of  origin  of  the  macrophages  and  of  the  monocytes  with  the  hope  that 
some  distinctive  features  might  be  observed  on  the  basis  of  which  the 
macrophages  and  monocytes  could  be  separated.  For  instance,  Naegeli 
claims  (1919,  etc.)  that  the  monocytes  are  of  myeloid  origin.  Therefore, 
differences  might  be  found  between  the  splenic  and  hepatic  veins,  draining 
the  chief  sites  of  origin  of  macrophages,  and  the  femoral  vein.  Niagara 
Blue  26  No.  8  was  autopsied  between  macrophage  showers  and  at  a  time 
when  the  monocyte  count  was  high  (13  %)  and  blood  from  all  these 
sources  was  analyzed.  The  blood  of  the  right  ventricle,  hepatic  vein  and 
iliac  vein  all  gave  a  monocyte  count  of  13  %.  The  results  then  threw  no 
light  on  a  possible  myeloid  derivation.  Failure  to  recognize  more  mono- 
cytes in  veins  from  marrow  might  be  due  to  the  slow  rate  of  production 
of  these  cells.  The  uniform  distribution  is  explained  from  the  fact  that 
normally  the  lungs  do  not  filter  the  cells.  The  same  count  in  all  vessels 
would  quickly  succeed  the  establishment  of  a  given  rate  of  production, 
and  the  femoral  vein  would  contain  approximately  the  same  content  of 
monocytes  as  any  other  vein.  No  case  was  examined  in  which  it  was 
definitely  known  that  the  count  of  the  monocytes  was  lower  in  the  arterial 
than  in  the  venous  blood,  so  there  was  no  reason  to  assume  greatly  in- 
creased activity  of  the  sources  of  origin  of  the  monocytes  to  account  for 
the  high  monocyte  counts,  as  was  necessary  in  the  cases  of  the  macro- 
phages where  it  was  known  that  these  cells  were  being  removed  from 


126  SIMPSON 

the  circulation  in  immense  numbers.  In  some  of  the  autopsies  the  femoral 
vein  showed  a  higher  monocyte  count  than  the  splenic  and  hepatic  veins 
and  right  ventricle,  and  at  others  a  lower  count.  Thus  in  India  Ink  No.  i 
the  femoral  vein  contained  24  %  monocytes.  The  other  sources  yielded 
21%,  29%,  and  17%.  In  Niagara  Blue  No.  256  the  femoral  vein  con- 
tained 4  %  monocytes.  The  other  veins  yielded  less  than  9  %  and  the 
right  ventricle  contained  5  %.  India  Ink  No.  2  gave  in  the  femoral  vein 
less  monocytes  than  in  the  other  veins  or  the  right  ventricle.  On  the 
whole,  however,  the  differences  were  all  insignificant  and  cannot  be  used 
to  support  any  source  of  origin  of  the  monocytes.  The  macrophages 
were  on  the  contrary  consistently  less  frequent  in  the  femoral  vein  than 
in  the  hepatic  and  splenic  veins. 

One  of  the  changes  in  the  blood  forming  organs  of  animals  used  in 
these  experiments  was  the  alteration  sometimes  observed  in  lymphatic 
tissue.  This  consisted  in  a  decrease  of  the  usual  lymphoid  cells  and  their 
replacement  by  cells  with  large,  clear  nuclei  (chromatin  in  fine  specks 
through  the  clear  nucleus  and  collected  at  the  nuclear  membrane)  and 
eosin  staining  cytoplasm.  These  cells  had  a  big  tendency  to  occur  in 
polynuclear  forms,  giant  cells. 

Niagara  Blue  26  No.  258  showed  in  the  lymph  nodes  an  increase  of 
these  large  cells  with  clear  nuclei.  In  this  case  the  center  of  the  follicle 
remained  fairly  normal. 

Niagara  Blue  26  No.  19.  The  centers  of  the  lymph  follicles  in  the 
spleen  were  largely  transformed  into  these  cells. 

Sodium  Lysalbinate  No.  22.  In  the  lymph  follicles  of  the  spleen  the 
large  clear  cells  constituted  about  two-thirds  of  the  center  of  the  follicle. 
In  the  lymph  nodes  probably  about  a  third  of  volume  of  the  follicle  has 
been  transformed  into  these  cells  (central  part). 

In  Lamp  Black  No.  i,  no  differences  were  noticed  in  the  lymph  nodes 
but  in  the  spleen  the  cells  of  the  periphery  of  the  lymph  follicle  seemed 
to  be  changing  to  this  cell  with  the  large,  clear  nuclei. 

In  Lamp  Black  No.  23  the  germ  centers  of  the  follicles  of  the  spleen 
showed  these  large  clear  cells  conspicuously,  due  to  the  fact  that  they 
were  phagocytic  in  this  case.  The  pulp  cords  and  sinusoids  (both  very 
evident  in  this  case  due  to  the  hyperaemia)  contained  these  cells  hi 
abundance. 

In  Gelatin  No.  24  the  centers  of  the  lymph  follicles  in  the  spleen  had 
been  largely  transformed  into  these  cells.  At  the  periphery  of  the  follicle 
it  was  difficult  to  tell  the  difference  between  the  outer  cells  of  the  follicle 
and  the  cells  of  the  pulp. 

The  animals  treated  chronically  with  India  Ink  were  uninteresting 
in  this  respect.  An  occasional  non-phagocytic  giant  cell  with  peripherally 
arranged  nuclei  was  seen  in  the  lymph  nodes.  In  India  Ink  No.  25,  how- 
ever, an  animal  which  received  one  large  dose  (10  c.c.  undiluted  ink)  and 
died  four  hours  later,  probably  as  the  result  of  the  heart  punctures, 
showed  an  extraordinary  condition  of  the  lymphatic  tissue.  In  the  lymph 
nodes  there  was  a  very  marked  transformation  of  the  lymphatic  tissue 
into  these  cells.  Giant  cells  were  very  numerous.  There  was  no  carbon 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD  127 

present  in  these  cells.   In  the  spleen  there  was  a  distinct  localization  of 
carbon-containing  cells  around  the  lymphatic  follicles. 

3.  Monocytes  and  macrophages  are  not  always  associated 
together  in  the  bodily  reactions  to  disease  or  experiment. 

An  enormous  overproduction  of  normal  blood  monocyte 
cells  may  occur  in  pathological  cases  (74.6%!  Schilling)  with- 
out the  appearance  in  the  blood  of  true  macrophage  cells. 
The  very  existence  of  pure  monocytoses  constitutes  one  of  the 
strongest  arguments  for  a  distinct  monocyte  strain.  Other- 
wise it  is  practically  inexplicable  why  some  of  the  cells  do  not 
undergo  conversion  into  macrophage  cells.  Further,  although 
the  monocytes  are  rather  suddenly  increased  or  decreased  in 
number,  they  never  exhibit  anything  approaching  the  shower 
phenomenon  shown  by  macrophages.  Again,  while  it  is  true 
that  in  general  the  monocytes  agree  with  the  macrophages  in 
their  responses,  yet  a  monocytosis  may  be  detected  before 
macrophages  appear  and  outlast  the  presence  of  the  latter 
cells  in  the  blood.  Indeed  the  behavior  of  the  monocytes  after 
a  macrophage  " shower"  has  passed  is  hard  to  reconcile  with 
the  identity  of  the  two  cell  types.  In  such  cases  the  macro- 
phages suddenly  disappear  from  circulation  and  the  mono- 
cytes remain  uncontaminated  with  a  mixture  of  macrophage 
and  the  recognition  of  typical  monocytes  is  not  obscured  with 
doubt.  None  of  the  perplexing  intermediate  stages  between 
monocyte  and  macrophage  are  present.  On  the  theory  of 
derivation  of  macrophage  from  monocyte  we  would  expect  the 
period  of  cessation  of  active  outpouring  of  these  cells  to  be 
represented  at  least  by  a  period  of  intermediate  or  partial 
conversion  to  the  large  cells.  The  intermediate  types  are  only 
seen  during  macrophage  showers  and  could  hence  easily  rep- 
resent young  macrophages  indistinguishable  though  they  are 
from  cells  which  we  might  otherwise  call  large  monocytes.  It 
might  be  urged  that  these  data  do  not  settle  the  question  of 
derivation  of  macrophages  from  monocyte  cells  but  only  on 
the  time  and  place  of  these  changes.  The  data  at  any  rate 
would  appear  to  negative  the  conception  of  a  widespread  and 
continuous  transformation  of  the  monocytes  in  the  free  cir- 
culation throughout  the  general  capillary  bed  of  the  body. 


128  SIMPSON 

The  following  data  were  obtained  on  the  relationship  of 
macrophages  and  monocytes  in  the  chronically  injected 
rabbits.* 

In  Gold  No.  4  the  monocyte  count  rose  to  10%  before  the  climax  of 
July  30. 

In  Sodium  Lysalbinate  No.  22,  the  monocyte  count  remained  between 
7  %  and  15  %  during  the  treatment  and  they  were  slightly  more  numerous 
before  and  after  the  showers  of  macrophages.  The  monocytes  rose  from 
10  %  to  15  %  before  the  climax  of  June  28. 

In  India  Ink  No.  3  the  monocyte  count  fell  from  15  %  to  7  %  after  the 
shower  of  March  31. 

In  India  Ink  No.  2,  April  19,  there  were  .8  %  macrophages  and  7  % 
monocytes  reported  in  the  right  ventricular  blood.  The  monocytes  were 
larger  than  usual  and  there  were  very  few  macrophages  large  enough  to 
put  them  beyond  question!  On  June  2,  the  monocytes  rose  to  10%  just 
before  the  shower.  During  the  onset  of  the  shower  cells  with  the  char- 
acteristics of  typical  monocytes  fell  to  2-3  %.  At  the  end  of  the  shower 
the  count  was  again  10%.  On  July  17,  the  monocytes  rose  from  15  %  to 
20  %  before  the  shower. 

In  Niagara  Blue  26  No.  8,  the  monocytes  were  18  %  at  the  end  of  the 
shower  of  April  10.  By  April  14  they  had  fallen  to  10  %.  (The  monocytes 
in  this  animal  never  went  below  10  %  during  the  treatment  of  this  animal.) 
At  the  end  of  the  climax  of  May  2,  the  monocyte  count  was  24  %  to  22  % 
in  the  right  ventricle  and  only  9  %  in  the  peripheral  blood  (May  4~May  6). 
By  May  7  the  monocyte  count  was  12  %-i4  %  in  the  right  ventricle  and 
7  %  in  peripheral  blood,  on  May  20  the  monocytes  were  15  %  in  the  right 
ventricle  and  4.9  %  in  the  peripheral  blood  (no  shower  of  macrophages). 

In  Niagara  Blue  26,  No.  256,  the  monocyte  count  never  sank  below 
6  %  during  the  treatment.  At  the  end  of  the  climax  of  June  27,  after  the 
macrophages  had  disappeared,  the  monocyte  count  continued  at  22  %  and 
15  %  during  the  next  two  days. 

In  Niagara  Blue  26,  No.  19,  the  monocytes  varied  between  5  %  and 
15  %  during  the  course  of  treatment.  At  the  end  of  the  climax  of  May 
24  the  count  was  15  %. 

There  were  only  exceptional  cases  in  which  the  attempts  made  to  dif- 
ferentiate between  the  monocytes  and  macrophages  were  sufficiently 
satisfactory  to  make  it  worth  while  to  report  the  percentages  of  these 
two  cell  types  during  the  macrophage  showers.  Such  a  separation  was 
made,  however,  in  Niagara  Blue  26  on  May  10.  Here  35  %  of  the  large 
mononuclear  cells  were  typical  monocytes  in  every  way,  while  35  %  were 
typical  macrophages.  This  may  merely  be  equivalent  to  saying  that  at 
times  there  were  less  disturbing  forms  seen  than  at  others.  However,  the 

*  The  normal  monocyte  count,  as  made  by  the  supra-vital  method  used  in 
all  the  counts  referred  to  here,  was  between  3%  and  5%.  The  blood  in 
which  the  counts  were  made  make  unless  otherwise  specified  was  from  the 
right  ventricle. 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD  I2Q 

data  given  above  indicate  that  there  are  some  significant  variations  in 
size  and  number  of  the  monocytes  to  be  observed  in  the  periods  between 
macrophage  showers. 

These  results  show  that  the  monocyte  counts  are  higher  in  chronically 
treated  animals,  and  that  just  before  the  macrophages  appear  in  great 
numbers,  the  monocytes  are  also  present  in  greater  numbers  than  at  other 
times.  Similarly,  after  the  shower  of  macrophages  ceases,  the  monocytes 
are  found  to  be  present  in  increased  numbers,  sometimes  very  high,  as  in 
the  case  of  Niagara  Blue  26  where  they  constituted  18-22  %  of  the  white 
cells  for  two  to  three  days  after  typical  macrophages  were  no  longer 
present.  These  monocytes  had  all  the  characteristics  of  the  monocytes 
of  normal  blood.  There  were  no  forms  present  to  be  confused  with  macro- 
phages. Though  the  monocytes  present  in  the  blood  at  one  time  are 
uniform  in  size  as  well  as  morphology  there  is  some  indication  that  at 
different  intervals  after  macrophage  showers  the  size  of  the  monocytes 
present  differs. 

Though  there  is  not  enough  evidence  to  make  it  conclusive  it  would 
appear  that  the  monocytes  decrease  in  size  after  the  climax.  In  the  case 
of  Niagara  Blue  2B,  No.  8,  during  one  climax  studied,  there  were  35  % 
definite  macrophages  and  35  %  of  cells  slightly  larger  than  the  usual 
monocytes  but  characteristic  in  every  other  way.  May  n,  the  macro- 
phages had  practically  dropped  out  (one  atypical  cell  in  a  count  of  340 
cells)  while  there  were  18%  monocytes.  These  monocytes  were  slightly 
smaller  than  those  of  the  day  before.  At  the  termination  of  another 
climax  in  this  animal,  May  4,  the  monocytes  constituted  24%  of  the 
white  cells  (macrophages  though  still  to  be  found  by  a  search  with  the 
low  power,  did  not  appear  in  a  usual  differential).  They  were  all  the 
large  type  of  monocyte,  but  none  gave  difficulty  in  separation  from 
macrophages,  i.e.,  by  the  usual  criteria  of  gradations  in  size,  type  of 
segregation  apparatus,  refractive  vacuoles,  etc.  On  May  6,  the  monocytes 
continued  high  (22%)  and  had  the  same  uniform  characteristics.  The 
peripheral  blood  (ear  vein)  was  examined  two  hours  after  the  heart 
puncture  which  yielded  this  count,  and  showed  only  9.1  %  monocytes. 
On  May  7,  the  monocytes  were  still  14.3  %  in  the  right  ventricle,  and 
only  7  %  in  the  peripheral  blood  taken  five  minutes  later.  On  still  another 
occasion  this  difference  in  monocyte  count  was  noticed  in  this  animal. 
On  May  20,  the  heart  puncture  showed  15  %  of  the  white  cells  were  mono- 
cytes. The  peripheral  blood  taken  one  hour  and  twenty-five  minutes 
later  showed  4.9  %  monocytes.  A  possible  explanation  (if  rapid  changes 
between  the  times  the  blood  was  taken  can  be  disregarded)  is  that  these 
cells  are  either  functionally  different  from  normal  monocytes  or  being 
larger  than  the  usual  monocytes  they  are  filtered  by  the  lung.  No  dif- 
ferences were  noted  in  normal  animals  between  right  and  left  ventricle 
monocyte  counts. 

Another  explanation  which  could  account  for  the  discrepancies  be- 
tween the  monocyte  count  on  the  two  sides  of  the  heart,  might  be  fur- 
nished by  the  plugged  condition  of  the  lung  capillaries.  The  differences 
in  the  counts  were  only  observed  for  a  short  interval  following  the 
macrophage  showers.  Under  these  conditions  even  normal  monocytes, 


130 


SIMPSON 

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132  SIMPSON 

which  are  the  largest  formed  elements  in  the  normal  blood,  might  be 
obstructed  when  passing  through  the  lungs. 

There  was  not  consistent  evidence  that  the  monocytes  decreased  in 
number  and  size  after  the  macrophage  showers.  In  Niagara  Blue  26, 
No.  19  and  No.  256  a  sudden  drop  of  the  macrophages  ensued  on  death 
from  88  and  58%  to  1.8  and  i%  respectively,  so  that  the  monocytes 
became  recognizable  within  a  short  time  after  the  macrophages  had  been 
present.  It  was  found  that  the  monocytes  were  present  in  only  compara- 
tively small  numbers  (5-10%,  a  number  corresponding  to  the  count 
characteristic  of  the  whole  period  of  chronic  treatment  in  these  animals). 
On  the  other  hand,  17-24  %  of  monocytes  were  found  under  similar  con- 
ditions *  in  India  Ink  No.  i.  In  this  case  there  was  no  evidence  of  filter- 
ing of  the  cells  even  at  this  short  interval  after  the  shower.  The  count 
was  high  in  both  right  and  left  ventricular  blood. 

If  one  assumes  that  the  monocytes  and  macrophages  are  independent 
cell  types,  then  it  must  be  supposed  the  monocytes  are  also  increased 
during  the  macrophage  showers  at  least  in  a  part  of  the  cases.  If  a  par- 
allel increase  of  the  monocytes  is  supposed  to  occur  in  all  cases  then  one 
would  have  to  assume  that  the  same  factors  can  at  times  cause  the  ab- 
normal numbers  of  both  cells  to  disappear  from  the  circulation  in  order 
to  include  the  cases  of  Niagara  Blue  26,  No.  19  and  No.  256,  while  at 
other  times  the  monocytes  could  continue  to  be  poured  out  in  large  num- 
bers after  the  macrophages  had  disappeared. 

The  evidence  seems  more  convincing  for  the  supposition  that  an  in- 
crease of  monocytes  may  also  occur  under  the  conditions  which  produce 
the  macrophage  reaction. 

*  India  Ink  No.  i    8.57P.M.    LV    Macrophages  =    o%    Monocytes  =  16% 
9.04  P.M     RV  "  58  a      cannot  be 

separated  from 
macrophages 

Autopsy 10.00  P.M.    LV  o  22 

RV  "  o  17 

Hepatic  vein "  o  21 

Femoral  vein o  24 

Splenic  vein i  20 

An  answer  can  be  given  to  the  other  arguments  for  the 
identity  of  macrophage  and  monocyte  cells.  Mcjunkin's 
oxydase  and  phagocytosis  tests  are  inadequate.  Apart  from 
the  fact  that  the  common  positive  oxydase  reaction  claimed 
by  Mcjunkin  is  denied  for  both  cells  by  some  writers  (Schil- 
ling, Schlener),  we  can  say  that  both  of  these  reactions  — 
oxydase  and  phagocytosis  —  are  shared  with  other  cells  and 
tissues.  They  can  hence  no  more  be  regarded  as  establishing 
the  identity  of  these  two  cell  types  than  an  identity  for  instance 
of  macrophage  and  myeloid  cells. 


MACROPHAGES  IN  THE  CIRCULATING  BLOOD  133 

In  the  monocytoses  produced  by  bacterial  and  other  causes 
Mcjunkin  has  not  shown  —  except  by  his  term  "endothelial 
leucocyte" — that  endothelium  is  especially  involved.  Indeed 
it  must  be  pointed  out  that  he  has  not  produced  or  studied 
cases  in  which  true  circulating  macrophages  were  seen  —  such 
as  the  present  study  reports.  We  are  confident  of  this  because 
of  (i)  his  descriptions  of  these  cells  which  seem  typical  mono- 
cytes;  (2)  his  studies  were  made  on  peripheral  blood,  and  (3) 
the  experimental  treatment  occupied  too  short  a  time  interval, 
the  most  chronic  dosage  employed  by  him  consisting  of  six 
doses  of  carbon  given  in  seven  days.  The  lamp  black  suspen- 
sion was  the  same  as  that  employed  in  this  work.  Such  a 
treatment  in  the  rabbit  would  not  even  yield  macrophages  in 
any  great  number  in  the  venous  blood  of  the  heart  and  they 
would  not  be  seen  in  the  peripheral  blood  even  with  much 
longer  treatment. 

The  present  study  then  can  lay  claim  to  being  the  first  in 
which  any  considerable  experimental  production  of  circulating 
macrophages  has  been  attained  and  has  furnished  the  first 
opportunity  of  investigating  both  the  correspondence  and  the 
differences  in  these  cell  types.  Furthermore  there  has  been 
employed  here  a  reliable  method  for  the  recognition  of  mono- 
cytes  —  the  supra- vital  stain  with  Janus  Green  and  Neutral 
Red.  These  methods  have,  however,  only  emphasized  the 
more  the  close  biological  kinship  of  monocyte  and  macro- 
phage  cell  types. 

By  them  a  remarkable  similarity  in  morphology  and  vital 
staining  reactions  is  apparent  as  well  as  the  existence  during 
macrophage  showers  of  typical  intermediate  cell  forms. 
Furthermore,  a  short  time  following  these  showers,  the  mono- 
cytes  were  partially  filtered  out  by  the  lungs  just  as  the  ma- 
crophages are  almost  completely  filtered  by  the  pulmonary 
circulation.  Such  evidence  points  convincingly  at  any  rate 
to  a  kinship  in  these  two  types  of  cell,  a  kinship  further  ex- 
emplified by  their  common  response  in  many  clinical  and 
experimental  conditions.  To  conclude  from  this  that  the  cells 
are  identical  would  appear  unjustifiably  simple.  At  any  rate 


134  SIMPSON 

until  more  crucial  or  specific  tests  are  discovered  this  position 
would  seem  indefensible.  With  it  we  should  have  above  all 
to  explain  how  it  is  that  the  monocytes  do  not  normally  or 
at  least  more  frequently  undergo  any  of  the  steps  toward 
macrophage  transformation,  or  how  in  special  circumstances 
the  mother  tissue  of  these  cells  can  be  considered  to  proliferate 
at  the  same  time  to  produce  two  cell  types  as  a  result  of  stimu- 
lation by  the  same  substance. 

SUMMARY  AND  CONCLUSIONS 

I.  Methods  of  producing  circulating  macrophages. 

1.  Macrophages  can  be  produced  by  the  chronic  intrave- 
nous injections  of   the  following:    (a)  Colloidal  dyes,  e.g., 
Niagara  Blue  26 :  Lithium  Carmine. 

(b)  Larger  colloids  and  suspensoids,  e.  g.,  Red  gold  in  sodium 
lysalbinate  solution:  India  ink:  Lamp  black  in  gelatin  solution. 

(c)  Certain  proteins  or  split  products  of  proteins :  Gelatin : 
Sodium  lysalbinate. 

2.  The  effectiveness  of  the  members  of  Group  (b)  may  have 
been  due  to  the  presence  of  the  protective  colloids  of  Group  (c). 

3.  It  has  been  found  impossible  to  determine  a  stimulating 
action  peculiar  to  the  higher  colloids  and  the  suspensoids, 
because  of  their  instability  in  the  absence  of  the  protective 
colloids. 

4.  The  only  point  that  could  be  found  in  common  between 
the  three  kinds  of  stimulating  agents  by  which  the  macro- 
phages were  obtained  in  the  circulating  blood,  was  that  all  the 
solutions  injected  contained  some  substance  in  the  colloidal 
state. 

II.  The  " shower  phenomenon."    When  the  macrophages 
appear  in  the  circulating  blood, 

1.  They  are  distributed  unevenly  in  the  body,  with  none  or 
very  few  in  the  peripheral  blood,  more  in  the  venous  blood 
of  the  heart,  and  most  in  the  venous  blood  from  the  haemo- 
poietic  organs,  especially  the  liver  and  the  spleen. 

2.  The  difference  in  the  macrophage  content  of  the  two 
ventricles  is  very  striking,  (a)  Due  to  the  great  numbers  of 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD  135 

the  cells  poured  out  of  the  haemopoietic  organs,  and  (b)  be- 
cause of  the  practically  complete  removal  of  the  macrophages 
by  the  lungs. 

3.  The  macrophages  are  not  present  constantly  in  the  blood 
of  chronically  injected  animals  but  appear  in  " showers";  i.e., 
in  chronically  treated  animals  a  time  is  finally  reached  when 
the  animal  responds  to  each  succeeding  injection  by  pouring 
great  numbers  of  cells  into  the  general  circulation. 

4.  Judging  from  the  lack  of  included  foreign  matter  which 
had  been  injected,  the  macrophages  were  produced  greatly  in 
excess  of  the  number  needed  to  handle  the  foreign  matter. 

5.  Marked  changes  in  the  platelet  content  and  the  coagula- 
bility of  the  blood  are  associated  with  the  shower  phenomenon. 

III.  The  relationships  of  the  macrophages  to  the  normal 
mononuclear  cells  of  the  blood. 

1.  Monocytes  and  macrophages  can  be  separated  from  the 
lymphocytes  by  means  of  a  Supra- vital  stain  of  the  living  cells. 

2.  Monocytes  and  macrophages  when  present  simultane- 
ously in  large  numbers  can  not  be  distinguished  by  the  Oxy- 
dase,  Giemsa,  Supra-vital,  or  other  tests,  though  it  may  be 
possible  to  separate  them  by  their  ability  to  stain  with  the 
vital  benzidine  dyes. 

3.  These  two  cells  may  at  times  act  independently;   they 
nevertheless  participate  in  common  in  many  of  their  biological 
reactions  (i.e.,  they  appear  separately  in  the  clinical  mono- 
cytoses  reported  by  Schilling  and  in  the  experimental  mono- 
cytoses  reported  here,  while  both  cell  types  appear  to  be 
present  in  the  macrophage  showers  and  the  macrophagocytosis 
of  Schilling). 

4.  During  most  of  the  period,  even  of  long  or  chronic  treat- 
ment, macrophages  are  uncommon  in  the  main  circulatory 
current.   During  this  time,  however,  the  monocyte  group  can 
always  be  recognized  and  while  they  may  be  increased  in  num- 
ber (monocytosis)  they  retain  their  typical  or  normal  mor- 
phology. 

5.  During  macrophage  showers  the  monocytes  and  macro- 
phages are  related  not  only  by  staining  and  by  other  tests  but 
also  by  the  presence  of  many  intermediate  cell  types. 


136  SIMPSON 

6.  If  there  is  a  conversion  of  monocytes  into  macrophages 
it  at  any  rate  is  limited  in  time  and  space  and  does  not  occur 
indiscriminately  in  the  general  circulation. 

7.  The  evidence  at  present  available  indicates  a  close  bio- 
logical relationship,  but  there  is  not  a  proven  identity  of  these 
two  types  of  cell. 

(The  author  wishes  to  thank  Dr.  Herbert  M.  Evans,  whose  constant 
advice  and  help  made  possible  this  study.) 

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Moss,  W.  L.  &  Brown,  G.  L.    1911.   Variations  in  the  leucocyte  count 

in  normal  rabbits,  in  rabbits  following  the  injection  of  normal  horse 

serum,  and  during  a  cutaneous  anaphylactic  reaction.   Johns  Hopkins 

Hosp.  Bui.,  xxii,  258-268. 
Naegeli,  O.    1919.    Blutkrankheiten  und  Blutdiagnostik.    Berlin  and 

Leipzig,  3.  Aufl.,  Walter  de  Gruyter  &  Co.,  662  p.,  18  pi.  8°. 
Netousek,   M.     1913-14.    Endothelium  im  stromenden  Blute.    Folia 

Haematol.,  xvii,  407-412. 
Ottenberg,  R.  &  Rosenthal,  N.    1917.    A  new  and  simple  method  for 

counting  blood  platelets.  Jour.  Amer.  Med.  Assoc.,  Ixix,  999. 
Pappenheim,  A.    1900.    Von  den  gegenseitigen  Beziehungen  der  ver- 

scheidenen  farblosen  Blutzellen  zu  einander.  Virch.  Arch.,  elk,  40-85. 

1907.    Einige  Bemerkungen  liber  Methoden  und  Ergebnisse  der 

sog.   Vitalfarbung  an  den  Erythrozyten.   Folia  Haematol.,  iv,  Suppl. 

46-53- 

Patella,  V.  1909.  Der  endotheliale  Ursprung  der  Mononuklearen  des 
Blutes.  Folia  Haematol.,  vii,  218. 

Permar,  H.  H.  1920.  An  experimental  study  of  the  mononuclear  phag- 
ocytes of  the  lung.  Jour.  Med.  Research,  xlii,  9-32. 

1920.    The  development  of  the  mononuclear  phagocytes  of  the 

lung.   Ibid.,  147-162. 


140  SIMPSON 

Permar,  H.  H.     1920.     The  migration  and  fate  of  the  mononuclear 

phagocyte  of  the  lung.    Ibid.,  209-225. 
Reschad,  H.  &  Schilling-Torgau,  V.    1913.    Ueber  eine  neue  Leukamie 

durch   echte   uebergangsformen    (Splenozyten-Leukamie),    und   ihre 

Bedeutung  fiir  die  Selbststandigkeit  dieser  Zellen.    Miinchen.  med. 

Wchnschr.,  Ix,  1891-1894. 
Rieux,  J.   1910.  Du  grand  mononucleaire  du  sang  et  des  variations  dans 

les  divers  etats  pathologiques.    Folia  Haematol.,  x,  209-224. 
Rosenthal,  N.  1917.  Studies  on  the  oxidase  reaction  of  the  cells  in  normal 

and  leukemic  blood.   Arch.  Int.  Med.,  xx,  184-197. 
Rosin,   H.   &  Bibergeil,   E.     1902.     Ergebnisse    vitaler    Blutfarbung. 

Deutsch.  med.  Wchnschr.,  xxviii,  41. 

1904.  Ueber  vitale  Blutfarbung  und  deren  Ergegnisse  bei  Erythro- 

cyten  und  Blutplattchen.   Zeitschr.  f.  klin.  Med.,  liv,  197-222. 

1004.   Das  Verhalten  der  Leukocyten  bei  den  vitalen  Blutfarbung. 

Arch.  f.  path.  Anat.,  clxxviii,  478-504. 

Rowley,  M.  W.  1907.  A  fatal  anemia  with  enormous  numbers  of  cir- 
culating phagocytes.  Jour.  Exper.  Med.,  x,  78-97. 

Sacerdotti  C.  1893.  Intorno  alle  piastrine  del  sangue.  Arch,  per  le 
scienze  med.,  xvii,  35-55. 

1900.   Erythrocyten  und  Blutplattchen.  Anat  Anz.,  xvii,  249-253. 

1900.    Globules  rouges  et  plaquettes.    Arch.  ital.  de  Biol.,  xxxiii, 

344- 

1908.    Pouvoir  hemolytique  naturel  et  soustractions  sanguines. 

Arch.  ital.  de  Biol.,  i,  197-209. 

1911.    Anaphylaxie,  leucocytes,  plaquettes  et  serum  antiplaquet- 

tique.  Arch.  ital.  de  Biol.,  Ivi,  1-16. 

Schilling,  V.  1916.  Ueber  das  Leukozytenbild  bei  Variola  vera.  Miinchen. 
med.  Wchnschr.,  Ixiii,  154-156. 

1919.   Ueber  hochgradige  Monozyten  mit  makrophagen  bei  Endo- 
carditis und  ueber  die  Herkunft  der  grossen  mononuklearen.  Zeitechr. 
f.  klin.  Med.,  Ixxxviii. 

1920.     Ueber   Technik   der   Leukozytenuntersuchung   und   ihre 


praktischen  Erfolge.   Berl.  klin.  Wchnschr.,  Ivii,  895-897. 
Schlenner,  F.    1912.    Ueber  Technik  der  Oxydase  Reaktion   und  ihr 

Verhalten  an  Monozyten.   Deutsch.  med.  Wchnschr.,  xlvii,  6-7. 
Schultze,  W.  H.    1909.    Die  Oxydasereaktion  an  Gewebsschnitten  und 

ihre  Bedeutung  fiir  Pathologie.   Beitr.  z.  path.  Anat.  (etc.),  xlv,  127- 

153- 

1917-  Zur  Technik  der  Oxydasereaktion  (Indophenolblausyn these). 

Centralbl.  f.  allg.  Path.  u.  path.  Anat.,  xxviii,  8-n. 

1917.    Ueber  das  Paraphenylendiamin  hi  der  histologische  Farbe- 

technik  (kataly  tische  Farbung)  und  ihre  eine  neue  Schnellf  arbemethode 
der  Nervenmarkscheiden  am  Gefrierschnitt.    Ibid.,  257-260. 

1910.   Weitere  Mitteilungen  iiber  Oxydasereaktionen  an  Geweb- 

schnitten.    Miinchen.  med.  Wchnschr.,  Ivii,  2171-2173. 


MACROPHAGES  IN  THE   CIRCULATING  BLOOD  141 

Simpson,  M.  E.  1921.  Unpublished  work  done  in  the  Anatomical  Lab- 
oratory of  the  University  of  California.  Preliminary  reports,  Anatomi- 
cal Record,  1921,  and  Univ.  Cal.  Publication  in  Anatomy,  Vol.  i,  Nos. 
i  and  2,  Vital  Staining  Human  Blood  with  special  reference  to  the 
separation  of  monocytes. 

Trotter,  R.  T.  1919.  Unpublished  work  done  in  the  Anatomical  Labora- 
tory of  the  University  of  California. 

Van  Nuys,  F.  1907.  An  Extraordinary  blood:  presence  of  atypical 
phagocytic  cells.  Boston  Med.  and  Surg.  Jour.,  clvi,  390. 

Winkler,  F.  1907.  Der  Nachweis  von  Oxydase  in  den  Leukozyten  mittels 
der  Dimethylparaphenylendiamin-Alphanaphthol  Reaktion.  Folia 
Haematol.,  iv,  323  and  v,  17. 

1912.  Die  Farbung  der  Leukozytengranula  mit  Sudan  und  Alpha- 

naphthol.  Folia  Haematol.,  xiv,  23-25. 


DESCRIPTION  OF  PLATES  I-V 

Magnification  in  all  figures  was  2000  diameters. 

With  the  exception  of  figure  i  all  drawings  are  of  living  cells. 

PLATE  I.  Fig.  i.  —  Jenner-Giemsa.  Macrophage  obtained  from  the 
right  ventricle  of  a  chronically  injected  rabbit.  The  drawing  rep- 
resents an  average  sized  cell.  The  nucleus  shows  the  cluster  of  pale 
blue  staining  nucleoli.  The  cytoplasm  contains  the  numerous  fine 
granules.  The  granules  are  stained  purple  by  the  Giemsa. 

Figs.  2-8.  Illustrations  of  cells  which  had  phagocytised  injected  ma- 
terials. 

Fig.  2.  —  A  macrophage  obtained  from  a  right  ventricular  puncture  of 
India  Ink  No.  3  on  March  5,  1921,  i  hour  after  the  injection  of  20  c.c. 
of  diluted  (1/50)  ink.  Brilliant  Cresyl  Blue  counterstain.  This 
illustrates  the  complete  absence  of  a  segregation  apparatus  which 
sometimes  occurs  in  the  macrophages.  (The  cell  was  near  another 
macrophage  which  was  loaded  with  the  counterstain.)  A  very  small 
amount  of  carbon  has  been  phagocytised. 

Fig.  3.  —  A  macrophage  obtained  from  the  left  ventricle  (?)  of  Lamp 
Black  and  citrate  No.  6  on  February  22,  1921.  The  animal  at  this 
time  had  received  34.2  c.c.  Lamp  Black  and  74.2  c.c.  sodium  citrate. 
The  last  dose  had  been  given  24  hours  previous  to  the  heart  punc- 
ture (4.2  c.c.  sodium  citrate,  5  c.c.  Lamp  Black).  The  segregation 
apparatus  was  preformed  and  stained  at  once  with  the  Neutral 
Red  counterstain.  The  segregation  apparatus  did  not  increase  in 
size  with  the  exception  of  a  few  of  the  small  granules  on  the  surface 
of  the  larger  structure. 

Fig.  4.  —  Small  macrophage  obtained  from  Lamp  Black  and  citrate 
on  March  4,  1921,  from  a  liver  puncture.  The  autopsy  occurred 
within  one  hour  of  the  last  injection  (4.7  c.c.  Lamp  Black  and  n  c.c. 
sodium  citrate).  Nile  Blue  Sulfate  (B  extra)  counterstain. 


142  SIMPSON 

Fig.  5.  —  Same  source  and  conditions  described  for  Fig.  5.  A  larger 
cell  which  has  also  phagocytised  an  erythrocyte. 

Fig.  6.  —  Macrophage  obtained  from  India  Ink  No.  2  at  autopsy  from 
the  splenic  vein.  (The  needle  was  inserted  into  the  vein,  pointing 
toward  the  organ  drained  by  the  vein  and  at  a  place  in  the  vein 
about  3-4  inch  from  the  point  the  vein  emerged  from  the  spleen,  and 
only  enough  blood  was  removed  to  make  four  coverslip  prepara- 
tions, so  that  probably  the  blood  obtained  had  been  present  in  the 
splenic  vein.)  The  last  injection  of  this  animal  (5  c.c.  undiluted  ink) 
had  occurred  2  days  previously.  The  blood  was  supravitally  stained 
with  Janus  Green  B  and  Neutral  Red.  The  cell  has  not  rounded  up 
as  the  macrophages  do  in  most  cases. 

Fig.  7.  —  Conditions  the  same  as  in  Fig.  6.  This  represents  the  medium 
size  of  the  macrophages  found  under  these  conditions.  The  amount 
of  carbon  included  is  also  moderate.  Some  cells  contained  so  much 
carbon  that  it  was  difficult  to  tell  they  were  cells  unless  the  nuclei 
could  be  seen  at  the  periphery  of  the  cells. 

Fig.  8.  —  Macrophage  obtained  from  the  autopsy  of  Carmine  No.  n, 
February  24,  1921.  The  cell  occurred  in  blood  from  an  hepatic 
puncture.  Last  Carmine  injection  was  made  one  hour  previous  to 
death.  Preparation  was  not  counterstained.  The  nucleus  has 
stained  with  the  Lithium"  Carmine  which  has  diffused  from  the 
granules. 

PLATE  II.    Variations  in  the  size,  segregation  apparatus,    refractive 
vacuole  content,  etc.,  in  macrophages. 

Fig.  9.  —  Macrophage  obtained  from  heart  puncture  of  Lamp  Black 
and  citrate  No.  6,  March  i,  1921.  (24  hours  since  last  carbon  injec- 
tion.) Brilliant  Cresyl  Blue  counterstain.  Other  cells  were  stained 
brilliantly  with  the  supra- vital  stain  but  this  cell  remained  free  of 
dye  even  after  hours. 

Fig.  10.  —  Macrophage  obtained  from  right  ventricle  puncture  of 
India  Ink  No.  3,  March  9,  1921.  Four  days  had  lapsed  since  the  last 
injection  (20  c.c.  1/50  India  ink).  Counterstained  with  Brilliant 
Cresyl  Blue  and  Sudan  III.  A  very  finely  granular  segregation  ap- 
paratus was  present  in  this  cell.  The  granules  increased  in  size. 
The  refractive  vacuoles,  it  will  be  seen,  did  not  concentrate  the 
Sudan  III. 

Fig.  ii.  —  Very  small  macrophage  obtained  from  right  ventricle  (?) 
of  Lithium  Carmine  No.  11,  February  22,  1921.  (February  19  the 
last  injection  had  been  given,  5  c.c.  i  per  cent  Lithium  Carmine.) 
The  cell  was  supra-vitally  stained  with  Brilliant  Cresyl  Blue.  The 
cell  differed  from  the  normal  mononuclears  of  the  blood  due  to  the 
presence  of  the  refractive  vacuoles,  and  by  the  fact  that  the  dye 
faded  from  the  cell  by  two  hours,  while  the  stain  in  other  mononu- 
clear  cells  was  still  increasing  in  intensity. 


MACROPHAGES   IN   THE   CIRCULATING  BLOOD  143 

Fig.  12. —  Macrophage  obtained  from  the  right  ventricle  of  Gold 
No.  4  on  June  30, 1921,  three  hours  after  the  injection  of  .12  gm.  of 
the  gold  preparation.  The  cell  was  counterstained  with  Neutral 
Red  and  Janus  Green  B.  The  cell  is  interesting  because  of  the  great 
number  of  the  refractive  vacuoles,  the  very  slight  segregation  ap- 
paratus (and  because  of  the  conspicuous  nucleoli). 

Fig.  13.  —  Macrophage  obtained  from  right  ventricle  of  India  Ink 
No.  3  on  March  3, 1921,  one  hour  after  the  injection  of  20  c.c.  of  1/50 
India  ink.  The  cell  is  supra-vitally  stained  with  Brilliant  Cresyl 
Blue.  The  cell  shows  an  average  development  of  the  segregation 
apparatus. 

Fig.  14.  —  Macrophage  obtained  from  a  puncture  of  the  splenic  pulp 
at  the  autopsy  of  Lithium  Carmine  No.  n,  February  24,  1921,  one 
hour  after  injection.  The  cell  has  been  counterstained  with  Brilliant 
Cresyl  Blue.  The  cell  contains  a  very  well  developed  segregation 
apparatus  beside  phagocytised  red  blood  cells. 

Fig.  15.  —  Same  source  and  conditions  as  in  Fig.  14.  The  cell  was  in- 
teresting because  it  had  been  wedged  in  among  red  blood  cells  for 
two  hours  and  fragments  of  red  cells  were  present  in  the  macrophage 
in  great  numbers.  The  largest  fragments  were  nearest  the  periphery 
of  the  ceU. 

PLATE  III.  Cell  forms  intermediate  between  typical  macrophage  and  the 
normal  mononuclear  cells  of  the  blood.  (Neutral  Red  and  Janus 
Green  counterstain.) 

Fig.  1 6.  —  Normal  lymphocyte  of  the  rabbit. 

Fig.  17.  —  Normal  lymphocyte  of  the  rabbit  (very  large). 

Fig.  18.  —  Normal  monocyte  of  the  rabbit. 

Fig.  19.  —  A  cell  difficult  to  distinguish  from  the  large  lymphocytes, 
except  for  the  complete  absence  of  segregation  apparatus.  (From 
Niagara  Blue  No.  256,  July  3,  1921.  Autopsy,  splenic  vein.) 

Fig.  20.  —  A  macrophage  entirely  like  that  represented  in  Fig.  19 
except  for  the  presence  of  the  segregation  apparatus.  (Same  source 
as  cell  in  Fig.  19.) 

PLATE  IV.  Cells  of  various  sizes  showing  different  types  of  segregation 
apparatus. 

Fig.  21.  —  Macrophage.  The  cell  was  obtained  from  the  right  ven- 
tricle of  Gelatin  No.  24,  June  27.  Known  to  be  a  macrophage  as 
it  was  one  of  twin  cells  which  had  just  resulted  from  division  of  a 
larger  macrophage. 

Fig.  22.  —  Probably  this  cell  is  a  small  macrophage.  It  was  obtained 
from  heart  puncture  blood  of  Niagara  Blue  2B  No.  256  on  June  27, 
1921. 

Fig.  23.  —  Macrophage  (?).  The  cell  was  obtained  from  the  right  ven- 
tricle of  Gelatin  No.  24,  June  27. 

Fig.  24.  —  Macrophage  or  monocyte  (?)  The  cell  was  obtained  from 
a  heart  puncture  of  Niagara  Blue  26  No.  256  on  June  27,  1921. 


144  SIMPSON 

Fig.  25.  —  Probably  a  macrophage  judging  from  the  irregularity  of 

the  segregation  apparatus.  The  cell  was  obtained  from  the  right  ven- 
tricle, Gelatin  No.  24,  June  28,  1921. 

Figs.  26,  27.   Macrophages.   Same  source  as  cell  represented  in  Fig.  25. 

Figs.  28,  29, 30.  Macrophages.  Obtained  from  right  ventricle  of  Sodium 
Lysalbinate  No.  22,  June  29,  1921. 

PLATE  V.    Drawings  of  living  cells  obtained  from  a  case  of  Libman's 
subacute  bacterial  endocarditis.* 

Supra-vitally  stained  with  Janus  Green  B  and  Neutral  Red. 

Fig.  31.  —  Macrophage  obtained  June  n,  1921,  at  10.35  A-M-  Draw- 
ing 11.00-11.30  A.M. 

Fig.  32.  —  Macrophage  or  Monocyte  obtained  June  21,1921,11.00  A.M. 
Blood  contained  11+  per  cent  of  cells  of  this  type.  Evidence  pointed 
to  these  cells  being  monocytes. 

Fig-  33-  —  Macrophage  obtained  May  16,  1921,  12.10  P.M.  Drawing 
made  from  12.15  to  2.30  P.M. 

Fig.  34.  —  Macrophage  obtained  June  n,  1921,  2  P.M.  Drawing  made 
2.30  to  3.30  P.M.  Dividing  cell.  Though  the  indentation  of  the 
nucleus  became  more  marked  during  observation,  the  process  did 
not  continue  past  the  point  illustrated  here.  Droplets  on  red  stain- 
ing strands  became  increasingly  larger  and  more  heavily  stained. 
The  strands  also  became  coarser  and  stained  a  very  deep  red.  The 
droplets  fused  into  irregular  bodies.  Various  stages  in  this  process 
can  be  seen  in  the  drawing. 

Figs  35,  36.  —  Macrophages  showing  phagocytic  function. 

Fig.  35.  —  Blood  obtained  June  21,  1921,  3.40  P.M.  Cell  is  in- 
jured. 

Fig.  36.  —  Blood  obtained  June  n,  1921.  Drawing  made  two  and 
one-half  hours  after  the  preparation  was  made.  Cell  represented  is 
injured.  The  nucleus  of  the  macrophage  is  staining  with  the  Janus 
Green  B  while  that  of  the  phagocytised  cell  is  taking  up  the  Neutral 
Red  more  than  it  is  the  Janus  Green. 

Fig.  37-  —  Macrophage.  Blood  obtained  May  16,  1921  at  3.15  P.M. 
The  cell  was  still  alive  though  the  preparation  was  old.  The  segre- 
gation apparatus  shows  the  late  accumulation  of  Janus  Green  in 
structures  originally  stained  only  with  Neutral  Red. 

*  C.  P. — University  of  California  Hospital,  1921. 


THE  JOURNAL  OF  MEDICAL  RESEARCH,  Vol.  XLIII,  No.  2,  April-May  1922 


JOURNAL  OF  MEDICAL  RESEARCH. 


VOL.  XLIII.  PLATE  I. 


4  * 


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Simpson 


Macro  phages 


JOURNAL  OF  MEDICAL  RESEARCH. 


VOL.  XLIII.  PLATE  II. 


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11 


14 


12 


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Macrophages 


JOURNAL  OF  MEDICAL  RESEARCH. 


VOL.  XLIII.  PLATE 


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JOURNAL  OF  MEDICAL  RESEARCH. 


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